Morel - PCR-RT-PCR in Situ-Light and Electron Microscopy MVS

PCR/RT-PCR in situ
LIGHT and ELECTRON MICROSCOPY
Methods in Visualization
Series Editor: Grard Morel
In Situ Hybridization in Light Microscopy Grard Morel and Annie Cavalier Visualization of Receptors: In Situ Applications of Radioligand Binding Emmanuel Moyse and Slavica M. Krantic Genome Visualization by Classic Methods in Light Microscopy Jean-Marie Exbrayat Imaging of Nucleic Acids and Quantitation in Photonic Microscopy Xavier Ronot and Yves Usson In Situ Hybridization in Electron Microscopy Grard Morel, Annie Cavalier, and Lynda Williams PCR/RT - PCR In Situ Light and Electron Microscopy Grard Morel and Mireille Raccurt
PCR/RT-PCR in situ
LIGHT and ELECTRON MICROSCOPY
Grard Morel, Ph.D., D.Sc. Mireille Raccurt, Ph.D.
CRC PR E S S
Boca Raton London New York Washington, D.C.
Library of Congress Cataloging-in-Publication Data

Morel, Grard. PCR/RT-PCR in situ : light and electron microscopy / Grard Morel, Mireille Raccurt. p. cm. -- (Methods of visualization) Includes bibliographical references and index. ISBN 0-8493-0041-X (alk. paper) 1. Microscopy--Technique. 2. Electron microscopy--Technique. 3. Polymerase chain reaction. 4. Reverse transcriptase. I. Raccurt, Mireille. II. Title. III. Series. QH207 .M67 2002 570.282--dc21
2002073649
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2003 by CRC Press LLC No claim to original U.S. Government works International Standard Book Number 0-8493-0041-X Library of Congress Card Number 2002073649 Printed in the United States of America 1 2 3 4 5 6 7 8 9 0 Printed on acid-free paper
SERIES PREFACE
Visualizing molecules inside organisms, tissues, or cells continues to be an exciting challenge for cell biologists. With new discoveries in physics, chemistry, immunology, pharmacology, molecular biology, analytical methods, etc., limits and possibilities are expanded, not only for older visualizing methods (photonic and electronic microscopy), but also for more recent methods (confocal and scanning tunneling microscopy). These visualization techniques have gained so much in specicity and sensitivity that many researchers are considering expansion from in-tube to in situ experiments. The application potentials are expanding not only in pathology applications but also in more restricted applications such as tri-dimensional structural analysis or functional genomics. This series addresses the need for information in this eld by presenting theoretical and technical information on a wide variety of related subjects: in situ techniques, visualization of structures, localization and interaction of molecules, and functional dynamism in vitro or in vivo. The tasks involved in developing these methods often deter researchers and students from using them. To overcome this, the techniques are presented with supporting materials such as governing principles, sample preparation, data analysis, and carefully selected protocols. Additionally, at every step we insert guidelines, comments, and pointers on ways to increase sensitivity and specicity, as well as to reduce background noise. Consistent throughout this series is an original two-column presentation with conceptual schematics, synthesizing tables, and useful comments that help the user to quickly locate protocols and identify limits of specic protocols within the parameter being investigated. The titles in this series are written by experts who provide to both newcomers and seasoned researchers a theoretical and practical approach to cellular biology and empower them with tools to develop or optimize protocols and to visualize their results. The series is useful to the experienced histologist as well as to the student confronting identication or analytical expression problems. It provides technical clues that could only be available through long-time research experience. Grard Morel, Ph.D. Series Editor
ACKNOWLEDGMENTS
The authors particularly thank their students Brice Ronsin, Sophie Recher, Elara Moudilou, Ccile Vivancos for invaluable help in developing the methodology. We also acknowledge Franoise de Billy for her expertise in plant biology and for the illustrations she provided. We are grateful to Professors Tomas Garcia-Caballero and Annie Cavalier for their help in this project. We also thank John Doherty for his excellent English translation. We thank the different corporations, Applied BioSystem, Hybaid, and MJ Research, for their material assistance and all the technical description provided. This work was carried out in the framework of the European Leonardo Da vinci project (Grant F/96/2/0958/PI/II.1.1.c/FPC), in association with Claude Bernard-Lyon 1 University.
VII
THE AUTHORS
Grard Morel, Ph.D., D.Sc., is a research director at the National Center of Scientic Research (CNRS), at University Claude Bernard-Lyon 1, Villeurbanne, France. Dr. Morel obtained his M.S. and Ph.D. degrees in 1973 and 1976, respectively, from the Department of Physiology of Claude Bernard University-Lyon 1. He was appointed an assistant of histology at the same university in 1974 and became Doctor of Science in 1980. He was appointed by CNRS in 1981 and became research director in 1989. Dr. Morel is a member of the American Endocrine Society, The International Society of Neuroendocrinology, The Society of Neuroscience, The American Society for Cell Biology, Socit Franaise des Microscopies, Socit de Biologie Cellulaire de France, and Socit de Neuroendocrinologie Exprimentale. He has been the recipient of research grants from the European Community, INSERM (National Institute of Health and Medical Research), La Ligue contre le Cancer, lARC (Association de Recherche contre le Cancer), Claude Bernard University, and private industry. Dr. Morels current major research interests include the internalization and cellular trafcking of ligand and receptor molecules (in particular, nuclear receptors for peptides), the regulation of gene expression, and paracrine interactions (low gene expression level of ligand in target tissue). Mireille Raccurt, Ph.D., is an engineer in biology in a laboratory of the CNRS (Molecular Physiology), at Claude Bernard-Lyon 1 University, Villeurbanne, France. She has worked in a number of different laboratories, forming the basis for her knowledge and expertise in the elds of cellular and molecular biology. She has published more than 20 papers in the eld of protein and nucleic acid detection from normal and pathological tissues. She has taught histology at the Lyon School of Medicine from 1976 to 1989. Moreover, she has regularly organized and taught the theory and practical courses of immunocytology, in situ hybridization, and PCR in training courses at light and electron microscopic levels in different European countries. She has organized six meetings or workshops. Her expertise and current research interests include the localization and regulation of hormone and receptor gene expressions, correlated with signaling molecules in normal and tumoral states. Most recently, she has become interested in extrapituitary expression of growth hormone in fetal and adult tissues and its regulation in mammary gland.
IX
CONTENTS
General Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abbreviations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 1 - General Principles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 2 - Preparation of Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 3 - Pretreatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 4 - Reverse Transcription (RT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 5 - Polymerase Chain Reation (PCR) . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 6 - Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 7 - Revelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 8 - Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 9 - Controls and Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 10 - Typical Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 11 - Examples of Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A - Equipment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B - Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
XIII XV 1 21 47 65 87 119 147 177 233 277 323 343 349 355 389 407
XI
General Introduction
GENERAL INTRODUCTION
Over the past decade, no new procedure in molecular biology has achieved such an exceptional degree of biotechnical acceptance as the polymerase chain reaction (PCR). This in vitro enzymatic amplication of particular genetic sequences is now a basic tool in experimental work, and has become a highly standardized process. More recently, improved enzymes, automated routines, and the possibility of carrying out the reaction on DNA chips have opened entirely new applications for the method. In addition to its impact in laboratory research and medical diagnostics, PCR holds promise for signicant advances in quality control for agricultural products, and has become the most powerful weapon in the arsenal of forensic science. Powerful as PCR may be, however, it still involves the destruction of cells and tissue, and morphologists whose interests are associated with intact structures have remained unsatised. What they needed was a way to adapt PCR methods to undamaged cells or tissue sections to detect small numbers of copies of DNA or RNA in situ, while still preserving morphology. And in 1990, Haase et al. actually succeeded in amplifying lentiviral DNA in infected cells and detecting the amplication product by in situ hybridization. And so it was that in situ PCR came into being. This demonstrates that a mere technical innovation is sometimes all that is needed to give a fresh impetus to research. One of the rst successes achieved by the new technique was to conrm the relationship between HIV and AIDS. Initially, it had been found that only 1% of CD4 cells in the blood of asymptomatic seropositive subjects was infected by HIV, and it was difcult to see how such a small number of infectious particles could be responsible for such a serious condition. In 1992, however, several teams, including those of G. Nuovo and O. Bagasra, then that of Kominoth, began developing in situ PCR, which combined the amplifying power of the PCR with the ability of in situ hybridization to localize target sequences. Using this technique, it was demonstrated that in reality 30 to 40% of circulating CD4 cells were infected by HIV, thus making it clear that AIDS was essentially a viral disease, and underscoring the need for direct therapy against viral replication. In situ PCR and RT-PCR provided the rst means of detecting minute quantities of DNA or RNA in nondisrupted cells and tissue, with the possibility of subsequently detecting the amplied product at the site of origin. But if in situ PCR is to be successful, it requires considerable optimizationnot just of the PCR cycling but also of the xation step and tissue processing, the PCR tools and reagents used for the amplication, and the detection procedure. The objective of this book is to give scientists (morphologists, pathologists, or molecular biologists) the information they need about the basic approach to histological analysis, biochemistry of PCR, and to provide molecular biologists with a practical approach to histological analysis. Chapter 1 is a general presentation of in situ PCR/RT-PCR and the variants. The parameters for xation, tissue processing, and enzyme digestion are set out in Chapters 2 and 3. Reverse transcription (RT) and amplication techniques are described, with theoretical considerations and practical step-by-step guidance, in Chapters 4 and 5. Detection procedures are discussed in Chapters 6 and 7. And, given that in situ PCR can be combined with electron microscopy, the basic principles of these methods are presented in detail in Chapter 8. Numerous controls are, of course, needed to check for diffusion and potential causes of background, negative, or nonspecic signals. Finally, the causes of the false positives and false negatives associated with the different techniques are outlined in Chapter 9, with recommendations on how to avoid them. Chapter 10 provides guidelines that should help experimenters work out their own
XIII
General Introduction in situ PCR / RT-PCR protocol, although in the last analysis it is the empirical conditions themselves that dictate the precise details of any given protocol. Some examples of results are illustrated in Chapter 11. Methods for preparing the different reagents are given in the Appendices. Finally, it is the hope of the authors, who are themselves actively involved in the development of in situ PCR/RT-PCR, that the present work will provide practical solutions to some of the problems encountered by experimenters in the implementation of these complex, still-evolving techniques.
XIV
Abbreviations
ABBREVIATIONS
AEC ATP BCIP bp cDNA CTP DNA DAB dATP dCTP DEPC dGDP dGMP dGTP DNase dNTP DTT dUTP EDTA Fab (Fab)2 Fc FITC GTP Ig IgG kb kDa MM mRNA Mw NBT NTP Oligo (dT) PBS PCR PF RNA RNase rRNA 3-amino-9-ethylcarbazole adenosine triphosphate 5-bromo-4-chloro-3-indolyl phosphate base pair complementary deoxyribonucleic acid cytosine triphosphate deoxyribonucleic acid 3-diaminobenzidine tetrahydrochloride deoxy-adenosine triphosphate deoxy-cytosine triphosphate diethyl-pyrocarbonate deoxyguanosine-5-diphosphate deoxyguanosine-5-monophosphate deoxyguanosine 5-triphosphate deoxyribonuclease deoxynucleoside triphosphate dithiotreitol deoxyuridine-5-triphosphate ethylene diamine tetraacetic acid immunoglobulin fragment obtained by proteolysis (papaine) immunoglobulin fragment obtained by proteolysis (pepsine) immunoglobulin fragment obtained by proteolysis (papaine) uorescein isothiocyanate guanosine triphosphate immunoglobulin immunoglobulin G kilobase kilodalton molecular mass messenger ribonucleic acid molecular weight nitroblue tetrazolium nucleoside triphosphate oligo-deoxythymidine phosphate buffer saline polymerase chain reaction paraformaldehyde ribonucleic acid ribonuclease ribosomic ribonucleic acid
XV
Abbreviations rt RT RT-PCR SSC TH Tm tRNA Tw U UDP UTP v/v w/v room temperature reverse transcription reverse transcriptionpolymerase chain reaction standard saline citrate hybridization temperature melting temperature transfer ribonucleic acid washing temperature unit (enzymatic activity) uridine-5-diphosphate uridine-5-triphosphate volume/volume weight/volume
XVI
CONTENTS
General Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abbreviations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 1 - General Principles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 2 - Preparation of Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 3 - Pretreatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 4 - Reverse Transcription (RT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 5 - Polymerase Chain Reaction (PCR) . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 6 - Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 7 - Revelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 8 - Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 9 - Controls and Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 10 - Typical Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 11 - Examples of Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A - Equipment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B - Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
XIII XV 1 21 47 65 87 119 147 177 233 277 323 343 349 355 389 407
XI
Chapter 1
General Principles
Contents
CONTENTS
1.1 Polymerase Chain Reaction (PCR) . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.1 1.1.2 1.1.3 Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Target Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.3.1 Primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.3.2 Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.3.3 Experimental Conditions . . . . . . . . . . . . . . . . . . . Advantages/Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.4.1 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.4.2 Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . The Main Types of PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5 5 6 6 7 7 8 8 8 8 9 10 10 11 12 12 12 12 13 13 13 14 15 15 15 15 16 16 17 19 19 19 19
1.1.4
1.1.5
1.2 Reverse Transcription (RT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.1 1.2.2 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Main Types of RT . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3 In Situ PCR/RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.1 Advantages/Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.1.1 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.1.2 Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . In Situ Amplication Methods. . . . . . . . . . . . . . . . . . . . . . .
1.3.2 1.4
Direct In Situ PCR/RT-PCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4.1.1 Amplication of a DNA Target Sequence (direct in situ PCR) . . . . . . . . . . . . . . . . . . . . . . . 1.4.1.2 Amplication of an RNA Target Sequence (direct in situ RT-PCR). . . . . . . . . . . . . . . . . . . . . Advantages/Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . 1.4.2.1 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4.2.2 Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.4.2
1.5
Indirect In Situ PCR/RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.1.1 Amplication of a DNA Target Sequence (indirect in situ PCR) . . . . . . . . . . . . . . . . . . . . . . 1.5.1.2 Amplication of an RNA Target Sequence (indirect in situ RT-PCR) . . . . . . . . . . . . . . . . . . . Advantages/Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.2.1 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.2.2 Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.5.2
1.6
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1
Polymerase Chain Reaction (PCR)
1.1 POLYMERASE CHAIN REACTION (PCR)

PCR is used for synthesizing and amplifying, in vitro, specic nucleotide sequences of which only a very small number of copies are present in a given biological sample. It was described and named by Mullis et al. in 1987 (although the general principle had previously been set out by Khorana et al.). Since then, the technique has been considerably improved and has found many new applications. In 1990, in particular, Haase et al. developed an in situ PCR technique which combined the advantages and disadvantages of in situ hybridization and PCR, using cell suspension, to study the DNA of Lentivirus. This chapter, which is theoretical in orientation, deals rst with the fundamental principles of liquid-phase PCR, then sets out the different in situ PCR techniques. Mullis, K. and Faloona, F., In: Methods in Enzymology, R. Wu, Ed., Academic Press, New York, 1987, Vol. 155, p. 335. Mullis was awarded the Nobel prize for chemistry in 1993. Kleppe, K. et al., J. Mol. Biol., 56, 341, 1971. Panet, A. and Khorana, H.G., J. Biol. Chem., 249, 5213, 1974. Haase, A. et al., Proc. Natl. Acad. Sci. U.S.A., 87, 4971, 1990.
1.1.1 Aim
The aim of PCR is to amplify a specic sequence (the target sequence) of deoxyribonucleic acid to make numerous copies of it. This in vitro amplication, which is exponential, results in the synthesis of millions of copies of a specic segment of DNA. This sequence is determined by its two ends, 5 and 3 (see Figure 1.1).
1.1.2 The Target Sequence

The target sequence is a known succession of nucleotides which is: Present on one of the strands of a doublestranded DNA molecule, or Part of the sequence of a single strand of DNA. This is the most common case (see Figure 1.1). If it comprises the entire sequence of the strand of DNA, it can be amplied by using a plasmid.
General Principles
S 5' 3'
S = target sequence
Sc = sequence complementary to S
3' Sc 5' 3' S B 3' A 5' Sc D C 5'
A = 3 end of the target sequence B = 5 end of the target sequence C = 5 end of the sequence complementary to the target sequence D = 3 end of the sequence complementary to the target sequence Figure 1.1 The target sequence.
1.1.3 Overview
After denaturation of the double-stranded DNA, PCR involves the hybridization and 3 extension of a pair of primers: An anti-sense primer A sense primer At the end of this cycle, two identical copies of the DNA target sequence will have been obtained. It is the repetition of this cycle that allows a large number of copies to be obtained. This is what is known as a polymerization chain reaction (PCR). 1.1.3.1 Primers See Section 5.3.2. See Figure 1.1. The anti-sense primer is complementary and anti-sense to A. It corresponds to sequence C. The sense primer is complementary and anti-sense to D. It corresponds to sequence B. Direction of the 3 5 polymerization. L In the case of single-stranded DNA, only the hybridization of the anti-sense primer can take place during the rst cycle. The sense primer hybridizes on the neosynthesized strand during the second cycle.
2 copies, where n = the number of cycles.

n
A primer hybridizes in a specic way to each of the two strands of DNA (S and Sc), thereby dening the sequence to be amplied.
5' 3' A 3' 5' D 3' 5' 3' 5'
Figure 1.2 Positions of the primers at the ends of the target sequence.
1.1 1.1.3.2 Cycle
The PCR reaction comprises a three-stage cycle: Denaturation Hybridization Extension
These are successive, consecutive stages. At 95C, the double helix of the DNA matrix linearizes, and the two strands separate out. The primers then hybridize to the ends of the two strands of the DNA target sequence. DNA polymerase, attached to the 3 ends of the primers, synthesizes the strands that are complementary to the two strands of DNA, in the presence of nucleotides, in a given reaction environment and precise temperature conditions. It is only at the end of the second cycle that the sequence of interest (a copy of the target sequence) is obtained (see Section 5.1). Each cycle doubles the number of copies of the target sequence and its complementary sequence.
These three stages together make up an individual cycle. The repetition of this cycle is the basis of the PCR. It involves multiplying the number of copies of the target sequence. 1.1.3.3 Experimental conditions
Like other techniques in molecular biology, the success of PCR techniques depends on a number of rules being followed. Working environment. The experimentation must be carried out in sterile, or noncontaminating, conditions. This necessitates: A room or bench reserved exclusively for PCR, if possible away from where the extractions take place The cleanliness of the equipment, pipettes, centrifuge, and thermocyclers The utilization of single-use consumables The utilization of lter cones that prevent the propagation of contaminants through the air Handling done in sterile conditions Verication of the quantity and quality of the DNA matrix. For this purpose, two types of densitometric measurement are carried out: At 260 nm, to calculate how much of the nucleic acid to be amplied is present in the sample At 280 nm, to calculate the quantity of contaminating proteins in the extract Division of the reagents. The reagents must be divided into aliquots as soon as possible. 7 Given its high yield, a PCR requires particular vigilance with regard to the possible presence of contaminating DNA, which in fact is responsible for the majority of false-positive results observed in PCR.
Gloves must be worn.
One unit of DO260 nm corresponds to 50 g of double-stranded DNA. The optimal DO260 nm/DO280 nm ratio is between 1.8 and 2. It indicates that the quality of the DNA matrix makes it suitable for PCR amplication.
General Principles Verication of the specicity of the two primers. Verication of the compatibility of the hybridization temperatures of the two primers. Verication thermocycler. Each primer must be complementary to just one sequence in the genome, as determined by comparison with data banks (see Section 5.3.2). If the hybridization is to be specic, it must be carried out at a temperature determined by the Tm of the primers. These Tms must be very close together (see Section 4.3.1.3). Stability and reproducibility of the temperatures at which the different stages are carried out (see Section 4.4.1). See Section 5.3.3. Kogan, S.C. et al., N. Engl. J. Med., 317, 985, 1987. It should be possible to change the temperature rapidly, reliably, and without manual intervention.
of
the
reliability
of
the
The quality of the enzyme. The availability of DNA polymerase that can withstand very high temperatures means that the limitations of the PCR due to its lack of stability no longer apply. This has led to the automation of the technique, and its growing popularity.
1.1.4 Advantages/Disadvantages
1.1.4.1 Advantages The sequences are dened by their 3 and 5 ends. The cycle takes just a matter of minutes. It is limited, however, by the efciency of the enzyme and the quantity of reagents. These sequences have to be known in order for the primers to be made. Amplication of particular sequence (see Section 1.1.5). Using a standard (see Section 1.1.5).
The amplication of specic sequences Rapidity Amplication proportional to the number of cycles Necessity to know only the sequences of the ends of the DNA sequence to be amplied Differential analysis Quantication possible 1.1.4.2 Disadvantages
The tissue and cell structure are destroyed. It is not possible to amplify RNA. The amount of amplication is limited. Absolute quantication is difcult.
The technique is carried out after the extraction of the DNA. Reverse transcripton is necessary (see Section 1.2 and Chapter 4). This is always less than the theoretical yield, and varies from one experiment to another. This is due to the yield and the standard.
1.1.5 The Main Types of PCR

There are several types of PCR: Symmetrical PCR The sequence of interest and its complementary sequence are amplied simultaneously. The amplication is exponential. If the number of cycles is n, the number of copies n will be 2 .
1.2 Asymmetrical PCR
Reverse Transcription (RT)
Nested PCR
Semiquantitative PCR
Quantitative PCR
Only the sequence of interest is amplied, but the amplied products are variable in size since the extension can stop either before or after the 5 end of the target sequence. The amplication follows an arithmetic progression. If the number of cycles is n, the number of copies will be 2n. This is a double PCR in which a pair of primers situated on the amplied fragment during the rst PCR makes possible a further amplication of a sequence, which is common but smaller than the amplied fragment, thus increasing the sensitivity of the PCR. This is used to estimate the number of copies of a sequence of interest that have been obtained, by comparison with the simultaneous amplication of a known synthesized sequence (i.e., the mimic), using the same reaction mixture. This is used to determine the number of copies of interest, by comparison with the simultaneous amplication of a range of dilutions of the mimic.
1.2 REVERSE TRANSCRIPTION (RT)

PCR can only be used for DNA amplication. For an RNA target sequence to be amplied, it must rst be turned into a complementary deoxyribonucleic acid (cDNA). A reverse transcription (RT) step is carried out, in the presence of: A primer, which may be of different types Triphosphate nucleotides A specic enzyme, i.e., reverse transcriptase The result is a DNA copy (i.e., a transcription of RNA into cDNA). This neosynthesized cDNA can then be amplied by PCR. Advantages Reverse transcription makes it possible to: Amplify a given type of RNA Amplify different types of RNA RNA cannot be amplied directly by PCR. Depending on the type of primer: all the different types of RNA, or just a certain number, can be transcribed into cDNA (see Section 4.3.1). An RNA solution is always highly unstable, and is best conserved in the form of cDNA. 9
See Chapter 4. See Section 4.3.1. See Section 4.3.3.
Stabilize a solution of different types of RNA by turning them into cDNA
General Principles Disadvantages This step is difcult to evaluate. The yield depends on a large number of factors. See Section 9.3. These factors are related to the primers, the reliability of the enzyme, etc. (see Chapter 4).
1.2.1 Overview
5' 3' AAAAA
RNA
5'
3' AAAAA
The hybridization of the primer
+ RT
5' RT
3' AAAAA
3'
5'
The extension of the primer in the presence of the enzyme (reverse transcriptase) and the deoxynucleotides (dATP, dCTP, dGTP, and dTTP) cDNA Figure 1.3 Reverse transcription (RT).
1.2.2 The Main Types of RT

There are three different types of RT, corresponding to the three types of primer: Poly (T) primers Random primers Specic primers These different types of RT can be combined with different PCRs to obtain a complete range of methods for the amplication of RNA. Symmetrical and asymmetrical PCR Nested PCR Semiquantitative and quantitative PCR Differential display PCR (ddPCR) All the different types of poly (A) RNA retrotranscribed into cDNA (see Section 4.3.1.1) Nonspecic transcriptions of many types of RNA into cDNA (see Section 4.3.1.2) The specic transcription of the target sequence (see Section 4.3.1.3) See Section 1.5.
Only the RNA that remains after differential hybridization will be retrotranscribed, then amplied.
10
1.3
In Situ PCR/RT-PCR
1.3 IN SITU PCR/RT-PCR

PCR is used in situ to visualize, in cell compartments, DNA or mRNA that is present only in a small number of copies. The results depend on the compromise that is established between Preserving The sequence of interest The success of this technique depends on the PCR rules, along with all the constraints linked to the preservation of morphology.
The tissue structure
The cell structure
This is present only in a small number of copies, which must be preserved in suitable conditions, i.e., without breaks or digestion by RNase (see Section 2.3). It is essential to link the detected expression to the typical organization of the organ. Fixation is an important step (see Chapter 2). This is necessary to identify the cell type responsible for the detected expression, and to limit the diffusion of the amplied product; the cell constitutes a veritable PCR chamber. PCR/RT-PCR is preceded by a set of pretreatments of the tissue or cells (see Chapter 3). These are indispensable to the penetration of the reagents. In most cases, the detection of the amplied products requires an immunocytological reaction that uses immunoglobulins of high molecular weight (see Chapter 7). This step is generally not a problem, since the tissue sections or cells are subjected to a certain number of chemical treatments (proteinase, DNase, etc.) and physical treatments (high temperature), which ensure the permeability of the membranes. Excessive membrane permeability leads to the diffusion of the amplied products, which is the main cause of false positives.
And facilitating the accessibility of The sequence of interest
The amplied products
While avoiding The diffusion of the amplied product
The tissue or cells are xed (see Chapter 2). Figure 1.4 Schematic representation of a xed cell.
11
General Principles Permeabilized cytoplasmic and nuclear membranes.
Accessible DNA or RNA target sequences Figure 1.5 A pretreated cell.
1.3.1.1 Advantages PCR or RT-PCR By immunocytological detection Characterization of the different positive and/or negative tissue components See Chapter 8.
This method can be used to amplify a given sequence of nucleic acid, either DNA or mRNA. It can be used to visualize the amplied product in situ. It can be used to identify the tissue structure. It can be adapted to the ultrastructural scale. 1.3.1.2 Disadvantages
Limited efciency of the amplication
Diffusion of the amplied products
The partial destruction of morphology
The necessity for specic equipment
The constraints imposed by the tissue environment (proteins, lipids, sugars) interfere with the PCR reagents. This theoretical question can only be resolved by the presence of internal controls (i.e., positive and negative cells on the same tissue section) and the presence of a positive control. This is due to the necessary pretreatments and to variations in temperature during the amplication cycles. A thermocycler that will take slides.
1.3.2 In Situ Amplication Methods

There are two main types: Direct reaction Indirect reaction which are applied to Tissue, or Cells in culture both in light microscopy and electron microscopy. 12 See Chapter 5. See Section 5.5.6. See Chapter 5. See Chapter 8. See Sections 5.3.1.3, 5.3.2.7, and 5.5.1.1. See Chapter 5.
1.4
Direct In Situ PCR/RT-PCR
1.4 DIRECT IN SITU PCR/RT-PCR

The aim of this approach is to obtain an amplied product by the incorporation of a label in the course of the amplication phase. This allows the amplied products to be detected by means of an immunocytological reaction. The label is either coupled to the deoxynucleotide triphosphates that are incorporated during the amplication process or present on the primers that dene this amplication. These labels are generally antigens or, in rare cases, radioactive isotopes (see Section 5.3).
1.4.1 Overview
1.4.1.1 Amplication of a DNA target sequence (direct in situ PCR) x Denaturation of the nucleic acids
y Amplication of the DNA target sequence in the presence of: Labeled primers , or Nucleotides carrying the label
z The DNA target sequences amplied and directly labeled
Figure 1.6 Direct in situ PCR. Either the amplied product is directly visible, through the emission of a radioactive label (Figure 1.7), or the incorporated antigenic label is detected immunocytologically (Figure 1.8).
See Chapter 7.
13
General Principles The macroautoradiographic lm or nuclear emulsion is apposed to the tissue sections or cells (see Section 7.3). The emitted radiation is recorded on a photographic support. The signal corresponds to the amplied DNA. Figure 1.7 Autoradiographic detection. The immunoglobulin attaches to the label, forming an antigen/antibody complex that is either directly visible (uorescent antibody) or detected by an enzymatic reaction (alkaline phosphatase or peroxidase) (see Section 7.2). Figure 1.8 Immunocytological detection of an incorporated antigen. 1.4.1.2 Amplication of an RNA target sequence (direct in situ RT-PCR) The poly (T) random or specic anti-sense primer hybridizes to the strand of RNA (see Chapter 4). The reverse transcriptase synthesizes cDNA (see Section 4.3.3).
Figure 1.9 Reverse transcription of RNA. x Amplication of a cDNA sequence in the presence of: Labeled primers , or Nucleotides carrying the label See Chapter 5.
y Direct marking of the amplied product
Figure 1.10 Amplication of retrotranscribed cDNA. 14
1.5 As with a direct PCR, the amplied product obtained by a direct RT-PCR is detected by autoradiography or an immunocytological reaction.
Indirect In Situ PCR/RT-PCR
Radioactive label (see Section 7.3). Antigenic label (see Section 7.2). The macroautoradiographic lm or nuclear emulsion is apposed to the tissue section or cells (see Section 7.3). The emitted radiation is recorded on a photographic support. The signal corresponds to the amplied RNA. Figure 1.11 Autoradiographic detection. Immunoglobulin attaches to the label to form an antigen/antibody complex that is directly visible (uorescent antibody) or detectable by an enzymatic reaction (alkaline phosphatase or peroxidase) (see Section 7.2). Figure 1.12 Immunocytological detection of an incorporated antigen.
1.4.2.1 Advantages Whether DNA (direct in situ PCR) or RNA (direct in situ RT-PCR). Hybridization is not necessary. All the neosynthesized products are labeled.
These methods are used to visualize amplied products directly. They are rapid. They are extremely sensitive. 1.4.2.2 Disadvantages
It is difcult to be sure that the reaction has taken place correctly. A large number of false positives may occur.
See Section 9.3. See Section 9.4.
1.5 INDIRECT IN SITU PCR/RT-PCR

The aim of this approach is the specic detection of neosynthesized amplied products by a further hybridization step. The probes carrying the label are either radioactive or antigenic, and form hybrids with the sequence of interest. These hybrids are visualized by autoradiography or detected by immunocytology. This hybridization uses two probes, each complementary to one of the strands of the amplied product (see Chapter 6). Unlike the direct amplication methods, the indirect methods mostly use a radioactive label, given that the risk of contamination is lower (see Chapter 11). See Chapter 7. 15
General Principles The specicity of the probes is essential to the characterization of the amplied products. A specic hybridization can compensate for a lower degree of specicity in the PCR. Their specicity must be total, and must not allow any nonspecic hybridization to take place in the optimal hybridization conditions. The length of the probes can be increased to the point where total specicity is achieved.
1.5.1 Overview
1.5.1.1 Amplication of a DNA target sequence (indirect in situ PCR) x Denaturation of the nucleic acids
y Amplication carried out in the presence of unmodied nucleotides or unlabeled primers (see Chapter 5)
z Amplied DNA sequences
{ Hybridization with a pair of labeled primers (see Chapter 5)
16
1.5
Indirect In Situ PCR/RT-PCR
| Labeled hybrids
} Immunocytological detection (antigenic label) See Section 7.2.
~ Autoradiographic detection (radioactive label) See Section 7.3.
Figure 1.13 Indirect in situ PCR. 1.5.1.2 Amplication of an RNA target sequence (indirect in situ RT-PCR) x Reverse transcription of mRNA (see Chapter 4)
y Amplication of retrotranscribed cDNA in the presence of unlabeled markers or unmodied nucleotides (see Chapter 5)
17
General Principles z Sequences of the amplied, unlabeled cDNA of interest
{ Hybridization with a pair of labeled primers (see Chapter 5)
| Labeled hybrids
} Autoradiographic detection (radioactive label) See Section 7.3.
~ Immunocytological detection (antigenic label) See Section 7.2.
Figure 1.14 Indirect in situ RT-PCR.
18
1.6
Conclusion
1.5.2.1 Advantages It is the specicity of the probes that ensures this. See Chapter 9. Low cost
The characterization of the amplied product. With this type of method, the specicity of the reaction is much higher and easier to check. The use of unlabeled primers and nucleotides. 1.5.2.2 Disadvantages
Indirect methods take longer than direct methods. They are also less sensitive.
See Chapter 5.
1.6 CONCLUSION
For a given sequence of nucleic acid, whether DNA or RNA, the choice between the different in situ amplication methods, as well as between visualization by an immunocytological reaction or by autoradiography, will depend on: The nature of the target sequence The concentration of this sequence The possibility of nonspecic reactions The characteristics of the different approaches can be summarized as follows: Target DNA RNA Technique PCR RT-PCR Method Direct Indirect Direct Indirect
The estimated number of copies per cell
Specicity + ++ + ++
Sensitivity ++ + ++ +
19
Chapter 2
Preparation of Samples
Contents
CONTENTS
2.1 Tissue Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1 Sampling Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2 Diagram of the Different Steps. . . . . . . . . . . . . . . . . . . . . 2.1.3 Fixation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3.2 Fixation Parameters . . . . . . . . . . . . . . . . . . . . . . 2.1.3.3 Fixatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3.3.1 Cross-Linking Fixatives . . . . . . . . . . 2.1.3.3.2 Precipitating Fixatives . . . . . . . . . . . 2.1.3.3.3 Fixative Mixtures . . . . . . . . . . . . . . . 2.1.3.4 Fixation Protocols . . . . . . . . . . . . . . . . . . . . . . . 2.1.4 Frozen Fixed Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.4.1 Cryogenic Agents. . . . . . . . . . . . . . . . . . . . . . . . 2.1.4.2 Freezing Method . . . . . . . . . . . . . . . . . . . . . . . . 2.1.4.2.1 Cryoprotection . . . . . . . . . . . . . . . . . 2.1.4.2.2 The Freezing Operation . . . . . . . . . . 2.1.4.2.3 Conservation . . . . . . . . . . . . . . . . . . 2.1.4.3 Frozen Sections . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.4.4 Advantages/Disadvantages . . . . . . . . . . . . . . . . 2.1.5 Fixed Parafn-Embedded Tissue . . . . . . . . . . . . . . . . . . . 2.1.5.1 2.1.5.2 2.1.5.3 2.1.5.4 Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Parafn Embedding . . . . . . . . . . . . . . . . . . . . . . Making Sections . . . . . . . . . . . . . . . . . . . . . . . . Advantages/Disadvantages . . . . . . . . . . . . . . . . 25 25 26 26 26 26 27 28 30 30 31 31 32 32 32 33 34 34 36 36 36 36 38 39 39 39 39 39 40 40 40 40 41
2.1.6 Nonxed Frozen Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.6.1 2.1.6.2 2.1.6.3 2.1.6.4 Freezing Protocols . . . . . . . . . . . . . . . . . . . . . . . Frozen Sections . . . . . . . . . . . . . . . . . . . . . . . . . Fixation of Frozen Sections . . . . . . . . . . . . . . . . Advantages/Disadvantages . . . . . . . . . . . . . . . .
2.2 Cultures/Cellular Smears . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1 Cell Origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1.1 The Different Possibilities . . . . . . . . . . . . . . . . . 2.2.1.2 Diagram of the Different Steps . . . . . . . . . . . . .
23
Preparation of Samples 2.2.2 Cell Cultures on Slides . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2.1 Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2.2 Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2.2.1 Cross-Linking Fixatives . . . . . . . . . . 2.2.2.2.2 Precipitating Fixatives . . . . . . . . . . . 2.2.2.2.3 Fixation Protocols . . . . . . . . . . . . . . 2.2.2.3 Advantages/Disadvantages . . . . . . . . . . . . . . . . 2.2.3 Cellular Smears. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3.1 Obtaining Samples . . . . . . . . . . . . . . . . . . . . . . 2.2.3.2 Fixation Protocol . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3.3 Advantages/Disadvantages . . . . . . . . . . . . . . . . 2.2.4 Cellular Pellets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.4.1 Obtaining Samples . . . . . . . . . . . . . . . . . . . . . . 2.2.4.2 Fixation Protocol . . . . . . . . . . . . . . . . . . . . . . . . 2.2.4.3 Advantages/Disadvantages . . . . . . . . . . . . . . . . 42 42 43 43 43 43 44 44 44 44 45 45 45 45 45
24
2.1
Tissue Samples
On what types of biological material can in situ PCR and RT-PCR be performed? What precautions have to be taken in collecting such material, and what are the xation conditions that must be adhered to? This chapter provides answers to such questions. Whatever the origin of the biological material, the objective of the methods presented here is to ensure that nucleic acids are preserved, along with tissue and cell morphology.
2.1 TISSUE SAMPLES

In situ PCR can be carried out on various types of sample, from complete organs to biopsy material, of either human or animal origin. In situ PCR is rarely carried out on vegetable tissue, which requires a specic type of preparation.
2.1.1 Sampling Conditions

The sampling conditions are strict, and must prevent all possibility of contamination by extraneous RNase or DNase. The sampling procedure must be rapid, and the subsequent operations carried out as soon as possible. These are: Gloves must be worn.
Sterilized or single-use equipment (asks, tubes, surgical instruments). This requirement is often not respected in the case of surgical resections carried out for the purpose of anatomopathological diagnosis. The expression of the results must take account of this fact. On the use of a cryostat to obtain sections, see Section 2.1.4. To obtain parafn-embedded tissue sections, see Section 2.1.5. Parafn has now been largely replaced by the synthetic embedding medium Paraplast . To obtain sections on a cryostat
Fixation, followed by: Freezing Parafn or Paraplast embedding
Freezing, without xation, followed by: Fixation on slides
25
Preparation of Samples
2.1.2 Diagram of the Different Steps
Tissue
Fixation
Freezing
Freezing
Paraffin embedding
Frozen sections
Frozen sections
Sections
Fixation
2.1.3 Fixation
2.1.3.1 Overview
Fixation is essential to the success of in situ PCR. Its purposes are: To shut down the cellular machinery, but in a state that is as close as possible to that of the living organism To preserve the organization of the tissue, along with the shape and volume of the cell To prevent autolysis or bacterial attacks To inactivate lysosomial enzymes and endogenous RNase It must be carried out quickly: In vivo Immediately after the obtaining of the fresh sample 2.1.3.2 Fixation parameters Fixation by perfusion Fixation by immersion The xation procedure must allow the in situ amplication of the DNA or RNA to be carried out in the place where it was synthesized.
The satisfactory xation of tissue depends on a number of factors:
26
2.1 The diffusion of a xative through tissue can be expressed by the formula: d = K t which shows that the penetration of the xative, d, is proportional to the square root of the xation time, t, with K the coefcient of diffusion of the particular tissue type, representing the depth, in millimeters, to which the xative penetrates in 1 hour. Temperature It is generally advisable to carry out the xation procedure at room temperature.
Tissue Samples
The coefcient of diffusion, K, depends on the tissue in question. It is generally measured on a gel, or on homogeneous tissue such as that of the liver. The values are generally lower for tissue than for gels, since the different constituents of tissue act as barriers to the penetration of the xative.
Fixative concentration Each xative has its own maximal efciency concentration. Higher or lower concentrations give rise to artefacts that are difcult to interpret. pH Fixatives are used in buffered solutions at a pH of between 7.2 and 7.4. The pH has an effect on the molecular conformation of various cell components, and too low or too high a pH can affect reactions between proteins and aldehydes. The osmolarity of the mixture (buffer + xative) must correspond to normal physiological osmotic pressure. Fixation time varies between 2 and 24 hours, depending on the size and origin of the biological samples.
While lower temperatures would reduce the effects of autolysis related to anoxia, they would also severely hinder the penetration of the xative, particularly in the case of aldehydes. If the concentration is too low, morphology will not be properly preserved. Too high a concentration leads to overxation, which reduces the accessibility of the nucleic acids due to an increase in protein DNA bonds. Phosphate or cacodylate buffers are the most common. It is important to check that the xative and the buffer do not interact with each other. The osmotic pressure can be adjusted by the addition of sodium chloride, or compounds such as sucrose. It should not exceed 24 hours. Beyond this there are problems due to overxation, and it becomes difcult to entirely eliminate the xative, even with prolonged washing.
2.1.3.3
Fixatives
Fixatives can be divided into three groups, according to their mode of action on proteins: Cross-linking xatives Aldehydes, e.g., formaldehyde and glutaraldehyde, which form intra- and intermolecular bridges by reacting with the amines in the lateral chains of the peptides. Alcohol and acetone, which are coagulants, cause proteins to precipitate. A number of different xatives can be combined in such a way that their advantages are cumulative and their disadvantages are canceled out. 27
Precipitating xatives Fixative mixtures
Preparation of Samples Most of these mixtures contain mercury salts, and are suitable for trichromic stainings, but should not be used for in situ PCR. 2.1.3.3.1 CROSS-LINKING FIXATIVES This is commercial formalin, which is supplied at a concentration of 37%. Note: The solution may contain stabilizing agents such as methanol (10 to 15%) or calcium. It is toxic if inhaled, or if it comes into contact with the skin or the mucous membranes. It should be handled under a ventilated hood.
Formaldehyde or formic aldehyde is a gas which, in aqueous solution, produces methylene glycol.
H C H O H OH C H OH
HCHO + 2H2O
OHCH2OH
Figure 2.1 Formula of the formaldehyde. The maximum number of these bridges occurs at a pH of between 7.5 and 8.
Mode of action: The rst stage in the reaction of this monoaldehyde with proteins involves the formation of amino-methylol groups, starting with their free amine groups, which then condense into methylene (CH2) bridges. Action on nucleic acids: Aldehyde groups react with nucleic acid template to form DNADNA, RNARNA, RNA/DNAhistone protein cross-links. Preparation: Neutral buffered formalin Saline formalin Advantages Rapid penetration
Most of these bridges are eliminated by postxative treatment of the tissue (washing, dehydration) and a suitable proteolytic digestion process. See Appendix B4.1. See Appendix B4.1. It is, however, advisable to use small fragments of tissue to obtain good-quality xation (the penetration speed is of the order of 1 mm/h). A satisfactory degree of conservation of cellular structures is indispensable if the diffusion of amplication products outside the cell, i.e., in the amplication medium, is to be avoided.
A high degree of conservation of proteins, which satises morphological requirements
The molecular network resulting from the methylene cross-links, which protects RNA from being broken down by RNase Disadvantages Prolonged xation by aldehydes causes breaks to occur in the DNA chain, and these may become extension sites through TaqDNA polymerase. 28
2.1 These conformational changes reduce the efciency of hybridization with homologous DNA. Paraformaldehyde This is a polymer of formaldehyde. It is prepared in a buffered solution without a stabilizing additive.
H C H O n n H OH C H OH
Tissue Samples
This is the most widely used xative for in situ PCR and RT-PCR techniques. This is its main advantage compared to formaldehyde. Otherwise, it has the same properties, advantages, and disadvantages as formaldehyde.
Figure 2.2 Formula of paraformaldehyde. Stock solution (see Appendix B4.3) Working solution (see Appendix B4.3) See above. The absence of precipitating xatives 1 month at 4C This is the best xative in terms of morphological conservation, but it should not be used for in situ PCR or RT-PCR techniques at concentrations higher than 0.05%.
Preparation: Paraformaldehyde 40% Paraformaldehyde 4% Advantages Identical to those of formaldehyde A pure solution, without preservatives Disadvantage A limited conservation time Glutaraldehyde
O C H CH2 CH2 CH2 O C H
Figure 2.3 Formula of glutaraldehyde.
Mode of action: It forms long-chain polymers, providing a high level of conservation of tissue structures. Preparation: A weak (0.0250.05%) solution of glutaraldehyde can be added to a 3.5% paraformaldehyde solution. Advantage It works rapidly: Glutaraldehyde at 1% xes proteins and peptides in 10 s, and free amino acids in 1 min. Disadvantage The cross-links, which make nucleic acids less accessible, are not reducible.
Working solution (see Appendix B4.3)
The lowest concentration that has a xative effect is 0.025%.
Cross-links considerably reduce the penetrability of tissue, even after intense proteasic digestion.
29
Preparation of Samples 2.1.3.3.2 PRECIPITATING FIXATIVES Alcohols conserve nucleic acids very well, but should not be used for the xation of tissue intended for in situ PCR due to the poor conservation of structures at all concentrations. Generally used only for xing frozen sections and cellular smears. It should denitely not be used for xing tissue.
Ethyl alcohol (CH3CH2OH) Methyl alcohol (CH3OH) Acetone
2.1.3.3.3
FIXATIVE MIXTURES This xative should therefore, in principle, not be used in hybridization or amplication techniques. Although this xative creates problems for those who want to make retrospective use of diagnosed clinical cases, it is the one that is most widely used by anatomopathologists in France. Some authors have, however, reported that undesirable effects are kept to a minimum if the xation time does not exceed 2 hours. It is strongly advised that this xative not be used for in situ PCR, since acetic acid hydrolyzes nucleic acids. Mercury salts can form complexes with nucleic acids, thus limiting the accessibility of the latter.
Bouins xative: A mixture of formalin, picric acid and acetic acid. It conserves tissue and cell morphology very well, but causes nucleic acids to break down and change their conformation, thus making them less accessible.
Carnoys xative: A mixture of ethyl alcohol, chloroform, and acetic acid. Zenkers xative: A mixture of acetic acid, mercury bichloride, potassium bichromate, and disodium sulfate.
Properties of Fixatives
Fixative Methanol Ethanol Acetone Formaldehyde Paraformaldehyde Glutaraldehyde Paraformaldehyde and glutaraldehyde Other mixtures
*
Penetration +++ +++ +++ ++ ++ + ++ ++
Conservation of Nucleic Acids +++ +++ ++ ++ ++ + ++ To be tested

*
Morphological Preservation + + + ++ ++ +++ +++ +++
By in situ hybridization with a poly (T) probe.
30
2.1 2.1.3.4 Fixation protocols
Tissue Samples
Fixation by immersion The samples should ideally not exceed 5 mm in diameter, and should immediately be immersed in a suitable xative.
Fixation time
424 h
rt 2 15 min
Rinses in a buffer solution (phosphate or cacodylate)
Fixation by perfusion, followed by xation by immersion. After the animal has been anesthetized, and the perfusion system put in place:
Rinse rapidly with a buffer solution. Inject the xative (generally paraformaldehyde in a phosphate buffer). 40 ml/min
This is the most commonly used xation method. The samples can be stored in a cassette bearing an identifying label until the embedding process is carried out. Paraformaldehyde and buffered formalin are the most commonly used xatives. See Section 3.1.3.1.3. This depends on the size of the samples. It is advisable to cut large samples again after 12 hours of xation to facilitate the penetration of the xative. rt = room temperature. Low temperatures considerably reduce the speed of penetration of aldehydes in particular. See Appendix B3. Theoretically, rinsing time should be proportional to xation time, but in the case of in situ PCR and RT-PCR prolonged rinsing can reduce tissue integrity, and should be avoided. This is recommended for the study of the central nervous system, and for delicate tissue. The heart is exposed by opening the rib cage. A canula is introduced into the aorta after the opening of the left ventricle. It is connected by a catheter to a peristaltic pump. An incision is made in the right ventricle so that the uid can circulate by perfusion. For a rat, around 50 ml of buffer is perfused at body temperature. Around 500 ml of xative in solution are necessary to perfuse a rat. The ow rate must be kept steady if serious tissue lesions are to be avoided.
Remove the organs to be studied rapidly. Immerse these organs, 12 h or fragments of organs, 4C, or in the same xative. rt
2.1.4 Frozen Fixed Tissue

After xation, biological samples can be frozen for conservation and storage purposes. Homogeneous freezing preserves nucleic acids in situ and ensures their structural conservation; these are the two factors that essentially determine the results of in situ PCR and RT-PCR. The hardening of the samples facilitates the subsequent cutting of sections. Visualization of amplied products in the conserved cellular compartment. 31
Preparation of Samples 2.1.4.1 Cryogenic agents Widely used as a cryogenic agent, given that it is easy to obtain and store. Use adequate containers, i.e., not airtight (risk of explosion). Storage containers must be kept in wellventilated surroundings so that the evaporating nitrogen gas does not reduce the relative percentage of oxygen in the surrounding air. The boiling temperature of liquid nitrogen is very close to that of its liquefaction, so that the immersion of a warm object causes it to boil. The resulting nitrogen gas forms an insulating layer around the sample (chelefaction phenomenon), thereby reducing the cryogenic properties of the liquid nitrogen. This avoids the insulation problem.
Liquid nitrogen (196C) Advantages Liquid nitrogen can be used for storing samples after freezing. It is not particularly dangerous.
Disadvantage Insulation effects
Isopentane (methyl-2-butane) cooled in liquid nitrogen (160C) Advantage Its temperature remains constant during the freezing procedure.
Dry ice (78C)
Below 160C, isopentane becomes viscous. A thin layer of viscous isopentane in the bottom of a receptacle dipped in liquid nitrogen will conrm that the latter is at the right temperature. This is a contact cryogen. It does not produce homogeneous freezing, and should be avoided. It can, however, be used for at samples.
2.1.4.2 2.1.4.2.1
Freezing method CRYOPROTECTION
Prior to freezing samples that have been xed and rinsed in a buffered solution, measures should be taken to limit damage caused by the formation of ice from water within cells. Water-soluble cryoprotectant can be used for this purpose.
Saccharose or -D-Glucopyranosyl -D-fructofuranosode Formula C12H22O11 32
For a cryoprotectant to be effective, it must diffuse rapidly, which means that it must be of low molecular weight. It also needs to be a non-electrolyte, so that it can penetrate the cell easily and mix with saline solutions. Chemically neutral
2.1 Mw Concentration 342.3 30% in 0.1 M phosphate buffer, pH 7.4 C3H8O3 92.09 510%
Tissue Samples
Glycerol Formula Mw Concentration Dimethylsulfoxide (DMSO) Formula Mw Concentration 2.1.4.2.2 THE FREEZING OPERATION
C2H6SO 78.13 515%
The concentration can be adjusted according to the tissue. If the latter contains a lot of water, successive baths of increasing concentration are advisable. Also nontoxic for most cells. In spite of its low membrane permeability, it is a very good cryoprotectant. It is perfectly miscible with water above its fusion temperature (+18C). Toxic. A good cryoprotectant. As an organic solvent it can, however, have an effect on cell membranes.
After coating with O.C.T. mounting medium (Tissue-Tek ) Place the sample in a rubber mold. Cover with O.C.T.
Highly recommended for samples of small size, which risk thawing out while being attached to the slide holder. This facilitates the cutting of the sections. x Coating medium y Sample z Embedding mold
1 2 3
Figure 2.4 Embedding O.C.T. mounting medium. Or in isopentane cooled in liquid nitrogen. x Sample y Isopentane z Liquid nitrogen
Dip in liquid nitrogen.
1 min 196C
Figure 2.5 Freezing in isopentane cooled in liquid nitrogen.
33
Preparation of Samples Remove the samples from the molds and quickly place them in cryotubes. Without coating: There are several possible techniques. Immersion of the samples in 1 min isopentane cooled in 160C liquid nitrogen Direct immersion in liquid nitrogen 1 min 196C
Freezing in vapor from liquid nitrogen after total immersion, to simulate progressive freezing Freezing by contact with dry ice
This is the most commonly used technique. At 160C, the risk of breaks is small, and the thermal exchanges lead to homogeneous freezing. Up to the disappearance of the insulation phenomenon The risk of breaks is high, especially for large samples. Samples can be placed in tubes, or wrapped in aluminum foil. In such conditions, they should be allowed to oat on the surface of the liquid nitrogen. This procedure cuts down the number of breaks. This technique should be considered a second best option.
2.1.4.2.3
CONSERVATION 196C 80C No time limitations At 80C the preservation will always be of shorter duration.
In liquid nitrogen In the freezer
2.1.4.3
Frozen sections RNase-free conditions must be observed. The sample must never thaw out during this step. If the sample was embedded in O.C.T. before freezing, it is sufcient to adhere the block to the chilled support with a thin layer of the mounting medium. This takes around 15 min with the cryostat closed. x Cryostat support y Frozen sample
Mounting the sample on the cryostat support Place the support on the arm of the cryostat (20C), and cover it with a layer of O.C.T. Rapidly position the sample according to the desired plane of the cut before covering it completely with the coating medium.
Wait for the temperature of the whole system (sample, support, and knife) to reach equilibrium ( 20C).
Figure 2.6 Mounting the sample on the cryostat support. 34
2.1 Making frozen sections The sections must be of regular thickness, between 7 and 10 m.
Tissue Samples
The sections are placed on slides that have been treated with 3-amino-propyl-triethoxysilane and sterilized in an oven for 3 hours at 180C.
The quality of the sections will have an effect on the nal results. The cryostat temperature should be adapted to the hardness of the sample. One, two, or three sections are placed on each slide. See Appendix A3. With the Perkin-Elmer thermocycler, specially made slides must be used. Cover disks and cover clips are used for amplication.
The proper spreading and adhesion of the section result from the difference in temperature between the section and the slide. x Frozen sample on support
1 3
y Knife
z Section
{ Transfer of the section on the slide
Figure 2.7 Production of frozen tissue sections. Drying At room temperature 14 h Complete dehydration is necessary for the adhesion of the sections to the slides and for conservation of the nucleic acids (RNase and DNase become active only in the presence of water). The slides remain usable for several months, or even years. It is important to leave the box at room temperature for around 2 h before opening it, to avoid the risk of condensation, and thus the rehydration of the sections. 35
In a vacuum jar Storage In hermetically sealed boxes, with a desiccant (silicagel)
1h 20C or 80C
Preparation of Samples Following step Proteolytic pretreatments 2.1.4.4 Advantages/disadvantages Optimal preservation of nucleic acids. Fixation by aldehydes allows tissue and cellular structure to be conserved, which is indispensable to obtaining satisfactory results. Risk of thawing
See Section 3.5.
Advantages Sensitivity Morphology
Disadvantages Storage conditions are strict, both for the samples and the slides. Frozen tissue is delicate, even when xed, and is less resistant than parafn-embedded tissue to drastic treatments such as proteolysis, or the high temperatures involved in PCR.
2.1.5 Fixed Parafn-Embedded Tissue

Parafn embedding hardens samples, which makes it easier to obtain histological sections. 2.1.5.1 Fixation See Section 3.1.3.3.1. Time depends on the size of the sample. The xation of animal samples by perfusion is not indispensable (see Appendix B4.1.1). Time depends on the size of the sample (see Appendix B4.3). It is generally considered that after 8 h of xation in Bouins xative the signal is very weak, and that after 15 h it has disappeared. Their presence can be checked by hybridization with a poly (T) probe.
The best results are obtained with aldehydic xatives. Neutral buffered formalin 224 h
Paraformaldehyde 4%
224 h
The amplication of innitesimal quantities of RNA or DNA allows the use of samples whose nucleic acids have been largely destroyed by the Bouins xative.
2.1.5.2
Parafn embedding
Figure 2.8 Parafn embedding cassette. 36
2.1 Washing samples in a buffer solution 23 15 min
Tissue Samples
All trace of xative must be eliminated. Most of the bridges created by aldehydes can be reduced by washing in an aqueous medium.
Dehydration in alcohol baths 12 h each of increasing strength: 70, 95, 100 Impregnation 12 h A bath of xylene or methyl cyclohexane (a solvent for parafn) A bath of solvent/parafn 12 h mixture (v/v) 56C Two parafn baths 14 h each 56C Embedding Place the sample, according to the desired orientation, in a mold covered with a thin layer of liquid parafn. The mold ts onto the lower part of the box in which the sample has been kept after xation; or
Parafn liquefaction temperature
After the combination of block and cover has been removed from the mold, it is attached directly to the microtome arm.
Figure 2.9 Positioning the sample in the parafn embedding mold. The use of Leukart bars on a at surface (e.g., an earthenware tile or a metal plate) Cover with liquid parafn. In this way, large samples (e.g., entire organs or embryos) can be embedded. x Filling the mold
y Putting the box in place
z Embedded sample Figure 2.10 Embedding a sample in parafn. Cool: On a refrigerating plate, or In cold water Storage of blocks The block comes out of the mold naturally after the completion of cooling. rt Conservation is indenite. 37
Preparation of Samples 2.1.5.3 Making sections The sections can be 7 m thick in the case of delicate structures (e.g., the spleen or the thymus) which are more sensitive to the subsequent treatments.
Preparing the sections Prepare the sections on a microtome, after the parafn block has been attached to the sample support arm. The thickness of the sections is maintained at 5 m.
x Cassette in the sample support arm y Knife
z Strip of parafn sections Figure 2.11 Production of parafn-embedded sections. Arranging the sections The sections are arranged on pretreated slides, then are spread hot on a drop of sterile distilled water. See Appendix A3.
x Parafn section y Sterile water z Pretreated sterile slide

2 1
Figure 2.12 Positioning a section on a slide. This method of spreading sections is the best way of ensuring RNase-free conditions. If the spreading is carried out in a water bath at 40C, the water must be sterile, and changed regularly. It is possible to perform PCR or RT-PCR with control on adjacent sections on the same slide, and thus in the same conditions. Figure 2.13 The positioning of three sections on a slide.
The spreading takes place on a heating plate maintained at a temperature of 40 to 45C.
Drying in an incubator at 37C. 12 h Storage of the slides in hermetically rt sealed boxes, with a desiccant. Following step Proteolytic pretreatments. 38
It is best to conserve samples in parafn blocks. See Section 3.5.
2.1 2.1.5.4 Advantages/disadvantages
Tissue Samples
Advantages This is the method that gives the best preservation of morphological structures. Parafn blocks and slides are easy to store. Conservation is more or less indenite. It is easy to make sections. Disadvantage Low sensitivity If it can be considered that better amplication compensates for loss of sensitivity, parafn embedding after xation by an aldehyde is the method of choice for in situ PCR and RT-PCR.
2.1.6 Nonxed Frozen Tissue

Freezing without xation involves the cryoxation of samples in their initial state, without any morphological or structural alteration. 2.1.6.1 Freezing protocols See Section 2.1.4.2.2. See Section 2.1.4.2.2. Fresh tissue is, in this case, frozen without cryoprotection.
With a coating of O.C.T. mounting medium. Freezing in liquid nitrogen. Without a coating Isopentane cooled in liquid nitrogen Vapor from liquid nitrogen Dry ice 2.1.6.2 Frozen sections
Frozen sections are obtained in the same way as xed frozen tissue. 2.1.6.3 Fixation of frozen sections
See Section 2.1.4.3.
Cutting frozen sections A set of 10 to 20 sections are cut and kept in the cryostat at 20C. Fixation The 10 to 20 slides are xed 15 min in a tray with buffered 4C paraformaldehyde at 4%.
See Appendix B4.3. Better results are obtained with xed sections. There is better adhesion and better conservation of cellular structures, which limits the migration of amplied products.
Rinsing in PBS buffer
2 10 min 4C
39
Preparation of Samples Dehydration in alcohol baths of increasing strength: 70, 95, 100 Drying Storage in hermetically sealed boxes, with a desiccant. Following step Proteolytic pretreatments 2.1.6.4 Advantages/disadvantages
5 min per bath rt 20C or 80C
Dry under a ventilated hood. Storage has no undesirable effects if the box is opened at room temperature (so that no condensation occurs), and the desiccant is changed each time the box is opened. See Section 3.5.
Advantages Ease of utilization Rapidity Optimal conservation of RNA
Nonxed samples are, however, more sensitive to temperature differences resulting from handling. This can be a major drawback with amplication techniques.
Disadvantage Morphology is poorly conserved.
2.2
CULTURES/CELLULAR SMEARS
In situ PCR and RT-PCR are easy to carry out on cells, whatever their initial presentation, in monolayer cultures, cell suspensions, or smears.
2.2.1
Cell Origin
RNase-free conditions are ensured by wearing gloves and making sure that the cellular cultures are kept in sterile surroundings.
Handling conditions are strict, and must provide protection against contamination by DNase or RNase. 2.2.1.1 The different possibilities
Using cells in suspension Cells are obtained either from cultures in suspension or after unsticking the cellular layer dissociation by enzymatic treatment and centrifugation.
Cultured cells on slides or coverslips Cell pellets
The original in situ PCR work was done on cells in supension in Eppendorf tubes containing a PCR reaction medium. After amplication, the cells are cytocentrifuged and treated for the detection of products amplied by in situ hybridization. These are monolayer cultures. These can be used in the same way as tissue samples.
40
2.2 Smears on slides Using a biological uid Obtaining cellular smears by spreading or cytocentrifugation on a slide. 2.2.1.2 Diagram of the different steps
Cultures/Cellular Smears
These can be made either by spreading or by cytocentrifugation (cytospins). Examples are cephalo-rachidian uid, blood, urine or ascitic uid.
Using a monolayer cellular culture
Cell culture
Culture on slide or cover-slide
Cell suspension
Fixation
Fixation
Freezing
Cryopreservation
Paraffin embedding
Frozen sections
Freezing
Paraffin sections
Frozen sections
41
Preparation of Samples Using a culture in suspension or a biological uid
Biological fluid
Cell suspension
Cell smear
Cytocentrifugation
Fixation
Fixation
Fixation
Freezing
Cryopreservation
Paraffin embedding
Frozen sections
Freezing
Paraffin sections
Frozen sections
2.2.2
2.2.2.1
Cell Cultures on Slides
Culture
Using a cell suspension obtained after the removal of cells by an enzymatic treatment: Trypsin at 0.25% in PBS 5 min Centrifugation 5 min 300600 g On slides treated with silane and placed in a petri dish
On slides with a culture chamber
See Appendix A3. The slides that are specially designed for the Perkin-Elmer thermocycler make the task easier. The use of three cover disks and three cover clips per slide makes it possible to test three different conditions. The slides must be made of glass (due to the high temperature of the denaturation cycle), and thick enough to hold the cover clips rmly.
Seeding at a concentration of 10 cells/30 ml medium

5
42
2.2 Checking the conuence of the cells Rinsing in a sterile 0.1 M 2 5 min phosphate buffer, or in PBS 2.2.2.2 Fixation
The aim is to eliminate all trace of the culture medium.
2.2.2.2.1 CROSS-LINKING FIXATIVES A number of different xatives can be used, but paraformaldehyde gives the best results. Paraformaldehyde at 4% in a 0.1 M phosphate buffer Paraformaldehyde at 2% in a 0.1 M phosphate buffer 2.2.2.2.2 PRECIPITATING FIXATIVES Concentration v/v 10/90 50/50 80/20 90/10
As for tissue samples, the xation must be immediate.
Mixture acetone/ethanol ethanol/methanol ethanol/methanol ethanol/water
The results obtained in these xation conditions are haphazard. Cell morphology is often poorly preserved, and the absence of a signal often indicates no more than the diffusion of PCR products, and/or other cellular components, outside the cells. Slides xed in this way can be stored in hermetically sealed boxes at 20C or 80C (see Section 3.2.2.2.6).
2.2.2.2.3
FIXATION PROTOCOLS
Single xation The slides are immediately xed either: In paraformaldehyde at 4% in a 0.1 M phosphate buffer 30 min 4C The xation time is very important: inadequate xation gives rise to the diffusion of amplied products, whereas excessive xation retards the penetration of the reagents. Some researchers have, however, had improved results after 1015 h of xation.
Or in paraformaldehyde at 2% in a 0.1 M phosphate buffer Double xation Good results have been obtained with the use of a precipitating xative, followed by postxation with paraformaldehyde at 2% in a 0.1 M phosphate buffer. Rinses Use a 0.1 M phosphate buffer or PBS. Dehydration Use ethanol baths of increasing strength: 70, 95, 100.
Several h 4C 5 min 4C 30 min 4C 2 5 min This depends on the buffer in which the xative was diluted. These dehydration and drying steps are important, as the presence of water could activate cellular RNase during storage. 43 An example is an alcohol/acetone mixture.
2 min per bath
Preparation of Samples Drying Dry under a ventilated hood. rt Storage The slides can be stored at 20C or 80C in hermetically sealed boxes, with a desiccant. Following steps Permeabilization of the cells Proteolytic pretreatments See Section 3.4. They can be carried out directly after xation and rinsing. The slides are then pretreated, dehydrated, dried, and stored at 20C.
2.2.2.3
Advantages/disadvantages Almost all the cells are adhesive.
Advantages It is easy to make cultures on slides. No complex handling is required. The natural spreading of the cells means that their structure is maintained for the purpose of in situ studies. Disadvantage Some cells are difcult to culture on glass slides. See above.
2.2.3 Cellular Smears

2.2.3.1 Obtaining samples
Starting with a cell suspension or a biological uid, the cells are either: Spread directly on the 50 l/slide pretreated slide Or projected by cytocentrifugation onto pretreated slides. In both cases the slides are dried. 2.2.3.2 Fixation protocol 10 min 16,000 g 5 min rt
To obtain a monolayer spread, the concentration of the suspension must not exceed 6 2 10 cells/ml. Cytospin
Fixation The slides are xed in the same way as monolayer cellular cultures. Dehydration They are dehydrated in alcohol baths of increasing strength. Storage These are in hermetically sealed boxes. 20C
See Section 3.2.2.2.3. See Section 3.2.2.2.3.
44
2.2 Following steps Permeabilization Proteolytic pretreatments 2.2.3.3 Advantages/disadvantages See Section 3.4.
Advantages Smears or cytospins are easy to carry out. No complex handling is required. Disadvantages Poor cellular adhesion Loss of morphology Suitable equipment is needed.
2.2.4 Cellular Pellets

2.2.4.1 Obtaining samples The cells can also be obtained by scraping from the bottom of a culture box. This is done after elimination of the supernatant.
Using a cell suspension or a biological uid, the cells are: Centrifuged 5 min Rinsed in a phosphate buffer, or in 5 min a culture medium without serum Centrifuged 5 min 6001000 g Fixed 2.2.4.2 Fixation protocol 1530 min 4C 3 5 min
Fixation of the pellet using a buffered xative, of which the most widely used are formalin and paraformaldehyde at 4% Rinsing in a buffer solution
See Appendix B4.3.
It is sometimes necessary to carry out a centrifugation between the different steps to maintain the pellet in place.
The pellet can then be considered as a tissue sample, and treated as such. Following steps Freezing, or Cryoprotection and freezing, or Parafn embedding 2.2.4.3 Advantages/disadvantages See Section 3.1.3. See Section 3.1.2.3.2. See Section 3.1.4.
Advantages The sections adhere well to the slide.
45
Preparation of Samples Access to the different cellular components does not require permeabilization. Several different cell types can be processed together. Disadvantages The cells lose their characteristic morphology. The procedure is demanding and painstaking.
Internal controls
46
Chapter 3
Pretreatments
Contents
CONTENTS
3.1 3.2 3.3 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dewaxing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1 3.3.2 Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 52 53 53 53 53 54 54 54 54 54 55 55 55 56 56 57 58 58 59 59 59 59 59 59 60 60 60 60 60 60 61 61 61
3.4 Permeabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1 3.4.2 3.4.3 Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5 Deproteinization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1 3.5.2 Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enzymatic Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2.1 Proteinase K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2.1.1 Parameters for Use. . . . . . . . . . . . . . . . 3.5.2.1.2 Protocols . . . . . . . . . . . . . . . . . . . . . . . 3.5.2.2 Other Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2.2.1 Parameters for Use. . . . . . . . . . . . . . . . 3.5.2.2.2 Protocols . . . . . . . . . . . . . . . . . . . . . . . Chemical Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5.3
3.6 Postxation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.1 3.6.2 Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.7 Optional Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.1 Acetylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.1.1 Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.1.2 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inhibition of Endogenous Enzymes . . . . . . . . . . . . . . . . . . . 3.7.2.1 Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.2.2 Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.2.1.1 Inhibition of Endogenous Alkaline Phosphatases . . . . . . . . . . . . . 3.7.2.1.2 Inhibition of Endogenous Peroxidases . . . . . . . . . . . . . . . . . . . . . Digestion by DNase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.3.1 Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.3.2 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.7.2
3.7.3
49
Pretreatments 3.8 Dehydration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8.1 3.8.2 3.9 Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 62 62 62 63
Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.10 Recapitulation of the Consequences of Pretreatments . . . . . . . . . . .
50
3.1 Whatever the biological material, its nature, or xation, pretreatment step(s) are indispensable to the permeabilization of cellular and/or tissue structures, and to making the target nucleic acid accessible to primers and enzymes in the amplication medium.
Overview
3.1 OVERVIEW
Pretreatments comprise four essential steps, which have to be adapted, checked, and optimized for each type of sample. The aim is to achieve a compromise between accessibility to the nucleic acids and an acceptable degree of morphological conservation. It must not be forgotten that the cell acts as a PCR chamber in which the amplied products must be maintained. Insufcient pretreatments will lead to false-negative results, while overaggressive pretreatments will produce irreversible alterations in the tissue, thereby giving rise to falsepositive results due to diffusion of the amplied products. Permeabilization by chemical agents Gloves must be worn; all the products must be RNase-free; the solutions must be prepared with DEPC water (see Appendix B1.2); and the equipment must be sterilized. Pretreatments are generally carried out on xed tissue or cells just before the PCR/ RT-PCR steps. They can also be carried out at the same time that frozen sections are being obtained from tissue, whether xed or not. The slides are then dehydrated and stored at 20C for amplication. This is especially important in the case of thick sections derived from parafn-embedded tissue. This partially eliminates proteins, and thereby reduces the bridges formed between cell proteins and nucleic acids by aldehyde xation. This stabilizes structures that have been weakened by permeabilization and deproteinization, and favors the adhesion of the sections to the slides. Because deproteinized sections are more easily broken down by RNase, this is an indispensable step, especially for pretreated slides stored at 20C. This is essential for thick sections. The destruction of genomic DNA guarantees specicity. This serves as a negative control of the in situ PCR reaction.
Deproteinization by enzymatic or chemical treatment Postxation
Dehydration
Destruction of targets DNase
51
Pretreatments As explained in the previous chapter, regardless of: The nature of the sample, or Its preparation The aim is to obtain xed tissue sections, cell smears, or suspensions. It is necessary to distinguish between the different types of pretreatment: Frozen tissue, xed either before or after freezing Parafn-embedded xed tissue Cellular fractions either on slides or in suspension Tissue or cells Fixation, freezing, embedding, or sections Fixation must be carried out before the pretreatment steps.
Frozen sections brought up to room temperature and rehydrated (see Section 2.1.4) Sections that have been carefully dewaxed (see Section 2.1.5) Considered as frozen sections (see Section 2.2) Permeabilization often sufcient in such cases
3.2 DIAGRAM OF THE DIFFERENT STEPS
52
3.4
Permeabilization
3.3 DEWAXING
3.3.1 Aim
Dewaxing consists of eliminating the embedding medium. It also allows the sections to be rehydrated. This step is carried out just before the permeabilizing treatments. A hydrophobic medium Sections cannot be stored after dewaxing.
3.3.2 Protocol
a. Dewax: Xylene or a substitute 3 10 min Although the risk of contamination in solvents is minimal, the baths must be changed regularly.
b. Rehydrate: Alcohol 100 Alcohol 95 Alcohol 70 NaCl 9 Following steps Permeabilization Deproteinization
5 min 5 min 5 min 5 min
Na ions maintain the nucleic acids in situ. Washing in water must be avoided.
+
3.4 PERMEABILIZATION
3.4.1 Aim
This step facilitates the penetration of reagents into the cells by the permeabilization of their membranes. This is a necessary step for thick sections of dense, tough tissue (e.g., muscle, heart, placenta) embedded in parafn.
Figure 3.1 Aspect of the cell after permeabilization.
53
Pretreatments
3.4.2 Reagents
Digitonin Detergents: For cells on slides or in suspension, the use of detergents provides sufcient permeabilization to make the use of proteolytic enzymes unnecessary. SDS
Sodium dodecylbenzenesulfonate Triton X100 Saponin Sarcosyl Nonidet P-40 (NP-40)
3.4.3 Protocols
All these detergents are prepared in a PBS or phosphate buffer. a. Dip the slides in one of the following baths: Digitonin, 0.05% 10 min rt Triton X-100, 0.1% 515 min rt Saponin, 0.1% 515 min rt Sarcosyl, 0.2% 515 min rt Nonidet P-40, 0.5% 1h at 4C b. After rinsing in a PBS or phosphate buffer, the preparations can be deproteinized. See Appendix B3.4
Room temperature
Used essentially for cell fractions on slides or in suspension
3.5 DEPROTEINIZATION
3.5.1 Aim
Incubation in detergent solutions is often insufcient to provide complete permeabilization of cells. In such cases proteolytic digestion is indispensable to the partial elimination of proteins. By breaking down DNA/DNA, and DNAhistones protein cross-linking, it makes nucleic acids more accessible to amplication products. Deproteinization is carried out either: By enzymatic treatment or By chemical treatment
Proteolysis is clearly tissue dependent, and this is a critical step, as the integrity of tissue and cells is to be preserved above all else.
Proteinase K, pronase, pepsin Acid hydrolysis
54
3.5
Deproteinization
Figure 3.2 Aspect of the cell after deproteinization.
3.5.2 Enzymatic Treatment

3.5.2.1 Proteinase K
Proteinase K is generally considered a selective protease for proteins associated with nucleic acids. When properly used, it is the enzyme that gives the best result in terms of morphological efcacy and preservation. 3.5.2.1.1 PARAMETERS FOR USE Enzymes differ considerably in terms of efciency, and tests have to be carried out when a different brand, or even a different batch of the same brand, is introduced.
Concentration The optimal concentration depends on the type of sample (cells, frozen tissue sections, and parafn-embedded tissue sections). Cytocentrifuged cells cultivated on slides or in suspension Frozen sections Parafn-embedded sections 1 g/ml 13 g/ml 310 g/ml
Moderate digestion (1 g/ml) gives good results. The optimal concentration depends on the thickness of the sections.
The concentration also depends on the nature of the tissue. The following values are given as a rough guide, for parafn-embedded tissue: Lung 13 g/ml This is one of the most delicate types of tissue.
Liver, intestine, kidney 3 g/ml Brain, pituitary 5 g/ml Muscle, heart 10 g/ml Dilution buffer Proteinase K is diluted in Tris-HCl/CaCl2 buffer (20 mM Tris, 2 mM CaCl2, pH 7.5). Temperature Optimal temperature 37C
See Appendix B3.7.2. This is a specic buffer, as calcium is the cofactor of proteinase K. The enzyme remains effective at lower temperatures. 55
Pretreatments Incubation time Incubation time varies.
5 to 30 min
Besides concentration, temperature and incubation time are the two parameters that modulate the action of proteinase K.
3.5.2.1.2
PROTOCOLS It is important not to open boxes of sections that have been stored at 20C until they have reached room temperature. See Appendix B2.19. See Appendix B3.4.1. It is best to use a thermostat-controlled water bath with a shaking mechanism.
a. Rehydrate dewaxed or frozen preparations stored at 20C: NaCl 9 5 min Phosphate buffer 0.1 M 5 min b. Place a tray containing Tris-HCl/CaCl2 buffer in a water bath. Dip the slides in the buffer when it has reached 37C. c. Enzymatic treatment: At the last moment, add proteinase K at 37C, and shake with the slide-tray to homogenize. Proteinase K 15 min 37C d. Stop the reaction by rinsing: In a Tris-HCl/CaCl2 buffer, and then in a phosphate buffer, or In a phosphate buffer containing 2 mg/ml of glycine, or By heat e. Postx: 2 min 5 min 2 5 min 2 min 95C
4% paraformaldehyde in a phosphate buffer f. Rinse: Phosphate buffer NaCl 9 Following step Postxation
5 min 3 5 min 2 min
This is an average time, which must be optimized for each type of sample. Residual proteolytic activity can deactivate Taq DNA polymerase. See Appendix B3.7.2. This change of buffer is meant to inhibit enzymatic action, and to eliminate NH2 groups from the Tris-HCl/CaCl2 buffer. Glycine is a proteinase K inhibitor. See Appendix B3.7.3. Use this for cell cultures on slides and cell smears. This step stabilizes structures weakened by deproteinization, and is particularly important for frozen tissue. Favors the maintenance of sections on slides for the amplication steps. To eliminate traces of xative Avoids the precipitation of phosphate in alcohols
3.5.2.2
Other enzymes These enzymes were widely used some years ago, but have now been abandoned because they are more difcult to use than proteinase K.
Other enzymes can also be used, e.g., pronase or pepsin.
56
3.5 3.5.2.2.1 PARAMETERS FOR USE
Deproteinization
Concentration As for proteinase K, the concentration depends on the nature of the sample, its xation, and the thickness of the section. Pronase Cytocentrifuged cells 500 g1 mg/ml cultivated on slides or in suspension Frozen sections 500 g1 mg/ml Parafn-embedded 14 mg/ml sections Pepsin
Tests must be carried out systematically.
The activity of this pH-dependent enzyme is maximal at pH 2, and is completely inhibited at pH 8. This is its most useful feature. Acid hydrolysis causes breaks in DNA.
Cytocentrifuged cells cultivated on slides or in suspension Frozen sections Parafn-embedded sections Dilution buffer Pronase TrisEDTA (TE) buffer PepsinHCl Temperature Pronase
1 mg/ml
1 mg/ml 14 mg/ml
pH 7.6 pH 25
Use 10 mM or 50 mM Tris (see Appendix B3.6, TE buffer). Its activity can be changed by increasing the pH (pepsin solution: 9.5 ml H2O, 0.5 ml 2 N HCl, and 20 mg pepsin). These enzymes are generally used at room temperature, or, to begin with, at a lower temperature. It is easier to control the incubation time factor than the temperature factor.
rt or 37C
Pepsin
Incubation time Pronase Cytocentrifuged cells 15 min cultivated on slides or in suspension Frozen sections 130 min Parafn-embedded sections 130 min Pepsin Cytocentrifuged cells cultivated on slides or in suspension Frozen sections
rt or 37C
This is a very tricky step.
This incubation time is always longer than for frozen tissue. These approximate values depend on the temperature and pH of the enzyme.
15 min
1560 min 57
Pretreatments Parafn-embedded sections 1560 min
3.5.2.2.2
PROTOCOLS See Appendix B3.6. Extemporaneous preparation (see Appendix B2.14.3). The glycine deactives the pronase. See Appendix B3.7.3. The sections can then be dehydrated in alcohol baths of increasing strength, after which they can be dried and stored. These are the classical conditions. This solution can be frozen, and will retain its activity for a week. The temperature and incubation time must be adapted to each type of sample.
1. Pronase a. Rinse the slides in TE buffer. b. Incubate the slides in the pronase solution. c. Rinse in TEglycine buffer (2 mg/ml). d. Rinse in NaCl 9. 5 min 5 min
2. Pepsin a. Rinse the slides in Tris-HCl buffer. b. Incubate the slides in the ~30 min pepsin solution (2 mg/ml in 0.2 N HCl, pH 5).
c. Rinse in Tris-HCl buffer, pH 8. Following step Postxation
5 min See Section 3.6
3.5.3 Chemical Treatment

Acid hydrolysis is an alternative to proteolytic digestion. Hydrochloric acid partly dissolves the bridges created by aldehyde xation, and its proteasic action is limited. a. Incubate slides in a solution of hydrochloric acid: HCl 0.050.2 N 10 min rt b. Rinse: Distilled H2O 25 min c. Dehydrate and dry. Following step Postxation This is currently very little used, because enzymatic treatments give better results. It causes breaks in nucleic acids.
See Section 3.6.
58
3.7
Optional Steps
3.6 POSTFIXATION
3.6.1 Aim
Postxation improves morphological preservation by stabilizing structures that have been modied by deproteinization. It also restores the adhesion of the sections to the slides for the following steps.
This is an indispensable step for frozen tissue sections and cell smears.
3.6.2 Protocols
There are two possibilities: Paraformaldehyde at 4% in a phosphate buffer Rinsing in a phosphate buffer Rinsing in NaCl 9 Alcohol 100 5 min 2 5 min 2 min 510 min To eliminate the xative To avoid phosphate precipitation in the alcohol Cold alcohol recommended for the postxation of cell cultures and smears An optional step (see Section 3.7.1) According to the revelation system used (see Section 3.7.2) An optional step (see Section 3.7.3) An optional step (see Sections 2.1.4.3 and 2.1.5.3)
Following steps Acetylation Inhibition of phosphatases and endogenous peroxidases Digestion by DNase Dehydration and drying
3.7 OPTIONAL STEPS

3.7.1 Acetylation
3.7.1.1 Aim The reaction also affects the nitrogenous bases of the nucleic acids, which can result in an overall reduction in the signal.
Acetylation turns the reactive amine group + ( NH 3 ) of the proteins into substitute amine groups (NHCOCH3), which are neutral. It thus reduces the background noise generated by electrostatic forces. This transformation is carried out by incubating the sections in acetic anhydride (CH3COCH3) in a triethanolamine buffer.
59
Pretreatments 3.7.1.2 Protocol
a. Dip the slides in a tray containing the triethanolamine buffer (0.1 M, pH 8), with magnetic shaking. b. Add the acetic anhydride, 0.25% with shaking. c. Incubate. d. Rinse: Phosphate buffer NaCl 9 e. Dehydrate. 1 min
This is the nal concentration. This compound is viscous, and dissolves only with shaking. The reaction begins immediately, and is complete in less than a minute. A single bath is enough to eliminate all trace of triethanolamine.
5 min 5 min
3.7.2 Inhibition of Endogenous Enzymes

3.7.2.1 Aim This step is to be carried out only if a positive signal is observed in the absence of amplication (without Taq DNA polymerase). Very often, variations in temperature inhibit endogenous activity. This step depends on the enzymatic system used for revelation. It is carried out either during the pretreatment of the samples or during the detection of the amplied product.
It is indispensable to eliminate all risk of false positives resulting from the revelation of endogenous enzymatic activity.
3.7.2.2 3.7.2.1.1
Protocols INHIBITION
OF ENDOGENOUS ALKALINE
PHOSPHATASES
Incubating the slides briey in a 20% aqueous acetic acid solution is an effective way of destroying hepatic alkaline phosphatases. However, intestinal and placental phosphatases are resistant to this treatment. a. Immerse the slides in a 15 s 20% acetic acid solution. 4C b. Rinse in a buffer solution. 3.7.2.1.2 INHIBITION OF ENDOGENOUS PEROXIDASES Hydrogen peroxide (H2O2) destroys endogenous peroxidases. a. Immerse the slides in a 0.1% sodium azide solution containing 0.3% H2O2. b. Rinse in a buffer solution. 60 10 min rt
Levamisol treatment is then necessary just before the detection of the amplied product (see Section 3.7.2.1.1 and Appendix B6.2.1.2).
If not, there is a risk that the nucleic acids may be hydrolyzed.
Hydrogen peroxide can be kept at 4C. It loses its effectiveness over time, and should be used within 3 months. See Appendix B6.2.1.3.
3.7
Optional Steps
3.7.3 Digestion by DNase

3.7.3.1 Aim
For the detection of mRNA by in situ RT-PCR, or for an in situ PCR control
DNase treatment destroys DNA (genomic, viral, or plasmid), sparing only target RNA.
This step must not be carried out until after proteolytic digestion, because the DNA must be accessible if the DNase is to be effective. This step is necessary when RT-PCR uses a pair of primers that are not specic to the RNA sought, as this involves a non-negligible risk of mismatching and nonspecic amplication.
Figure 3.3 Aspect of the cell after DNase.
In most cases, the primers are chosen on either side of an intron or a splicing zone so that the amplication is limited to the sequence being studied.
In such cases, this step is superuous. See Chapter 5.
3.7.3.2
Protocol 100 U/l The DNase must be of RNase-free quality. For RNase-rich cells, add 1000 U/ml of RNasin + 1 mM dithiothreitol (DTT) to the DNase solution. Some workers recommend much weaker concentrations of DNase, e.g., 1 U/l, and an incubation period of up to 18 h. See Appendix B1.2.
a. Prepare the DNase solution in a specic buffer (Tris-HCl 40 mM, pH 7.4; MgCl2 6 mM; CaCl2 2 mM).
b. Cover the sections with 20 to 30 l of this solution. c. Incubate in a moisture 1h chamber. 37C d. Rinse in a DNase buffer. e. Rinse in DEPC-treated sterile water. f. Dehydrate and dry. Following steps RT PCR
For in situ RT-PCR reaction, see Chapter 4. See Chapter 5.
61
Pretreatments
3.8 DEHYDRATION
3.8.1 Aim
Through the action of alcohols and air, the aqueous medium is eliminated from the section, to: Avoid any dilution of reagents in subsequent steps Protect the samples against the action of RNase and DNase Postxation by precipitating xatives
RNase and DNase are inactive in the absence of water.
3.8.2 Protocol
a. Dehydrate the preparations in alcohols of increasing strength: Alcohol 70, 95, 100 2 min/bath b. Dry: In a vacuum jar >30 min In air under a hood, >60 min protected against dust Following steps Storage at 20C in hermetically sealed boxes with a desiccant Reverse transcription Amplication
Room temperature.
The slides can be stored in this way, pending the PCR or RT-PCR step. See Chapter 4. See Chapter 5.
3.9 STORAGE
After dehydration, and the evaporation of the alcohol, the slides are stored in hermetically sealed boxes with a desiccant. Storage conditions rt 20C or 80C Storage time Several months
If the slides are stored at 20 or 80C, they must be allowed to return to room temperature before the box is opened, to avoid the reactivation of RNase. In favorable conditions, the storage time can be even longer.
62
3.10
Recapitulation of the Consequences of Pretreatments
3.10 RECAPITULATION OF THE CONSEQUENCES OF PRETREATMENTS

Preservation of Morphology ++ ++ + ++ ++
Accessibility of the Target Dewaxing Permeabilization Deproteinization Postxation Acetylation Inhibition of endogenous enzymatic activity Digestion by DNase Dehydration +++ ++ +++ ++
Intensity of the Signal + ++ +++ + + +++
Specicity of the Signal ++ + + ++ ++ ++ ++
Control
63
Chapter 4
Contents
CONTENTS
4.1 The Principle of Reverse Transcription. . . . . . . . . . . . . . . . . . . . . . 4.2 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 70 70 70 70 71 72 73 74 74 75 75 76 77 77 77 78 78 78 79 79 79 80 80 81 81 81 81 85 85 85
4.3 The Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1 Primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1.1 Poly (T) Primer. . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1.2 Random Primers . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1.3 Specic Primer . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2 Deoxynucleotide Triphosphates (dNTPs) . . . . . . . . . . . . . . 4.3.3 Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.3.1 AMV Reverse Transcriptase. . . . . . . . . . . . . . . . . 4.3.3.2 M-MLV Reverse Transcriptase. . . . . . . . . . . . . . . 4.3.3.3 Tth DNA Polymerase . . . . . . . . . . . . . . . . . . . . . . 4.3.3.4 Criteria of Choice . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Materials/Reagents/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.1 Thermocycler. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.1.1 Thermocycler for PCR, Either in Situ or in Liquid-Phase . . . . . . . . . . . . . . . . . . . . . . . . 4.4.1.2 Thermocyclers for in Situ PCR. . . . . . . . . . . . . . . 4.4.1.2.1 Apparatus with Slides Placed Vertically . . . . . . . . . . . . . . . . . 4.4.1.2.2 Apparatus with Slides Placed Horizontally . . . . . . . . . . . . . . Sealing Systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.2.1 The Cover Disk/Cover Clip System . . . . . . . . . 4.4.2.2 The Easyseal System . . . . . . . . . . . . . . . . . . . . Reagents/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4.2
4.4.3 4.5
Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.1 4.5.2 Reactive Medium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.2.1 Tissue Sections or Cells on Slides . . . . . . . . . . . . 4.5.2.1.1 Making a Hermetic Incubation Chamber . . . . . . . . . . . . . . 4.5.2.1.2 Incubation Temperature . . . . . . . . . . . 4.5.2.1.3 Duration of Incubation . . . . . . . . . . . . 4.5.2.1.4 Deactivation of the Enzyme . . . . . . . .
67
Reverse Transcription (RT) 4.5.2.2 Cells in Suspension . . . . . . . . . . . . . . . . . . . . . . . 4.5.2.2.1 Incubation Temperature . . . . . . . . . . . 4.5.2.2.2 Duration of Incubation . . . . . . . . . . . . 4.5.2.2.3 Deactivation of the Enzyme . . . . . . . . Washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.3.1 Tissue Sections or Cells on Slides . . . . . . . . . . . . 4.5.3.2 Cells in Suspension . . . . . . . . . . . . . . . . . . . . . . . 85 86 86 86 86 86 86
4.5.3
68
4.1
The Principle of Reverse Transcription
Reverse transcription (RT) is an indispensable step that turns messenger RNA (mRNA) into complementary DNA (cDNA) prior to the DNA polymerase chain reaction (PCR), which specifically amplies this newly-synthesized DNA. Thus, in situ RT-PCR is the most sensitive method for locating a specic, weakly expressed gene in a cell structure.
Gloves must be worn. RT must be carried out in RNase free conditions. All the equipment must be sterilized at 180C for 3 h, and the water used for the solutions must be sterile, or DEPC-treated (see Appendix B1.2).
4.1 THE PRINCIPLE OF REVERSE TRANSCRIPTION

RT requires: An RNA target matrix A primer, which may be various types Triphosphate deoxynucleotides An enzyme: reverse transcriptase A reaction medium
See Section 4.5.3.1. See Section 4.5.3.2. See Section 4.5.3.3. See Section 4.5.4.
Single-strand polyadenyl [Poly (A)] RNA.

5 1 AAAAAA 5 2 AAAAAA 5 3 3 TTTTTTT AAAAAA 3
Hybridization of the primer: x Poly (T) (TTTT)
y Nonspecic primer, either hexamer or nonamer () z Specic primer ()
3 5 3 5 AAAAAA 3 AAAAAA TTTTTTT 5 3 TTTTTTT 3 5
RT
Elongation by a reverse transcriptase (RT) using a poly (T) primer Obtaining cDNA Figure 4.1 The principle of reverse transcription. 69
RT
4.3 THE TOOLS
4.3.1 Primers
The choice of primer for RT depends on the chosen objective and the experimental approach, but also on the size and the conformation of the mRNA to be transcribed. 4.3.1.1 Poly (T) primer This is usable only with eukaryotes, because prokaryote RNA does not have a poly (A) end. Multiple labeling is possible. Several associations are then possible: Amplication by direct PCR with specic primers and incorporation of a labeled nucleotide and Indirect PCR using specic primers for the other sequence to be amplied, and detection by hybridization with labeled probes. Reverse transcription is either total or specic to the mRNA being sought. Choices are Poly (T) primer, random or specic primer.
This is an oligo-(dT) that, by complementarity, hybridizes with the poly (A) end of mRNA. Polymerization starts from this primer, and proceeds in the 5 3 direction. In this case, all the mRNA is reverse-transcribed into cDNA. It is then possible to amplify two different sequences on the same section, using specic primers.
70
4.3
The Tools
mRNA Hybridization of the poly (T) primer, and reverse transcription
Several different cDNA Figure 4.2 The principle of reverse transcription with a poly (T) primer. Characteristics It is generally 18 to 20 mers long. It must be puried to eliminate small poly (T) primers. Primer hybridization temperature Optimal enzyme temperature is 37 to 40C. 4.3.1.2 Random primers Hexamers and nonamers are mostly used to retrotranscribe long-strand RNA or sequences with secondary structures. Small poly (T) primers might then be considered as nonspecic primers that can hybridize with repeats of adenosine contained in mRNA.
These contain six or nine nucleotides, which hybridize in a random way with mRNA, as in random priming labeling method, which results in reverse transcription occurring at many points along the transcript.
mRNA Random hybridization of hexamers or nonamers, and reverse transcription
Numerous cDNA sequences, all different Figure 4.3 The principle of reverse transcription with random primers. Characteristics These are generally either hexamers or nonamers. The probability that such a sequence will encounter a complementary sequence on a 6 strand of RNA is, for a hexamer, 1/4096 (1/4 ) 9 and, for a nonamer, 1/262144 (1/4 ). Thus, the use of hexamers results in larger amounts of cDNA, while use of nonamers results in longer cDNA. These sequences hybridize with complementary or anti-sense sequences. 71
Their sequence is randomly chosen.
Reverse Transcription (RT) Hybridization temperature of random primers Incubation at 25C for 10 min is indispensable before incubation at the ideal temperature of the enzyme. 4.3.1.3 Specic primer This is the primer that generally gives the highest degree of specicity.
This facilitates the hybridization process.
In most cases, this is a synthetic oligonucleotide whose sequence is complementary and antisense to the mRNA that is to be amplied.
mRNA Hybridization of the specic primer, and reverse transcription cDNA Figure 4.4 The principle of reverse transcription with a specic primer. Characteristics Its sequence must be anti-sense and complementary to a unique sequence in the target sequence.
5
CAG AAT GCC CAG GCT GCG TTC TGC TTC TCA
As regards the cDNA strand with which it is meant to hybridize.
cDNA corresponding to the mRNA sequence Complementary anti-sense sequence Specic primer for a given sequence Figure 4.5 Determination of a specic primer sequence.
TTA CGG GTC CGT CGC AAG ACG AAG
GAA GCA GAA CGC AGC CTG GGC ATT
Its GC content must not exceed 50 to 55%. It is recommended that there be a G or C base, or, better still, a GC or CG base pair, at the 3 end, from which the neosynthesis initiates. It should be between 20 and 30 mers long.
Long sequences (>10 nucleotides) of poly (C), poly (G), or poly (GC) should be avoided. It is generally agreed that a strong bond at this end improves neosynthesis. Although an oligonucleotide of more than 18 mers is considered specic, it should be matched against a data bank to make sure that there is no homology with any sequence of the genome. This is a palindromic sequence.
The 5 end must not include a sequence that is complementary to a sequence situated at the 3 end, as this would entail a risk of autoligation. 72
4.3 Position The sequence must occur on an exon.
The Tools
This is often the anti-sense primer of the pair of primers that are necessary to the amplication (see Chapter 5).
I4 I4 3 E5
ATG 5 TATAA E1
I1 E2
I2 E3
I3 E4
I = Introns E = Exons, which together make up the cDNA Anti-sense primer Figure 4.6 The position of the specic primer in the structure of the gene.
Primer hybridization temperature For oligonucleotides, this temperature is based on their sequence, and is calculated as follows: For oligonucleotides with up to 20 bases Tm = 2 (A + T) + 4 (G + C) For larger oligonucleotides Tm = 16.6 log[Na ] + 0.41(% GC) + 81.5 % mismatches 67.5/base length 0.65 (% formamide)
+
The optimal RT reaction temperature, which is generally at Tm 8C, has to be conrmed experimentally. If the temperature is too high, the primer hybridizes only partly or not at all. Sequences other than the target RNA may then be converted into cDNA. Concentration Regardless of primer choice, the nal concentration of the primer must be optimized. An addition of 50 pmol of primer is recommended as a starting point for optimization.
This temperature is called the melting temperature (Tm). It corresponds to the formation of 50% of hybrids. Wallaces formula Where A, T, G, and C represent the corresponding numbers of nucleotides For a given oligonucleotide, the optimal Tm is generally given by the manufacturer. It is worked out by using the nearest neighbor formula (Breslauer). This formula takes into account the saline concentration of the reaction buffer, as well as the GC concentration. Given the optimal operating temperature of the enzyme (between 37 and 42C), the hybridization of the primer often takes place at low temperature (less than the suggested Tm). The use of Tth DNA polymerase (see Section 4.3.3.3), which works at 70C, can then be advantageous.
Final concentration is 1 M in a 50 l reaction volume.
4.3.2 Deoxynucleotide Triphosphates (dNTPs)

In reverse transcription, four triphosphate nucleotides are used at an equimolar concentration: dATP dTTP dCTP dGTP In the opposite case, the delity of the enzyme and the length of the transcribed cDNA can be affected. Deoxyadenosine Deoxythymidine Deoxycytosine Deoxyguanosine 73
4.3.3 Enzymes
The most frequently used enzymes are: AMV reverse transcriptase M-MLV reverse transcriptase Tth DNA polymerase Given that the manufacturers of these enzymes give information about their particular characteristics, this paragraph will present only their main features and modes of action. 4.3.3.1 AMV reverse transcriptase Avian myeloblastosis virus Moloney murine leukemia virus Thermostable polymerase of Thermus thermophilus The specics of the enzyme have to be taken into account in the RT step.
Origin AMV reverse transcriptase is extracted from the avian myeloblastosis virus. It is an holoenzyme with a molecular weight of 157 kDa. The mature dimer has different enzymatic activities: RNA-dependent DNA polymerase DNA-dependent DNA polymerase RNase H Quantity supplied The quantity supplied corresponds to a level of activity varying between 250 and 1000 U. The conservation buffer contains 50% glycerol to avoid freezing. Conservation In order for the enzyme to retain its maximum level of activity, it should in general be stored in aliquots at 20C.
The subunit is derived from the subunit by proteolysis.
A unit (U) is dened as the quantity of enzyme which, in 10 min at 37C, incorporates 1 nmol of insoluble dTTP in an acid medium, starting with an RNA [poly (A)] matrix and an oligo (dT) primer (see Section 4.3.1). Conservation time is extended by storage at 70C, and according to some suppliers an enzyme will remain stable for 24 months in such conditions. To avoid successive warmings, an isothermal refrigerated box should be used. This reaction requires a primer and Mg ions as cofactors.
2+
Principle of action AMV reverse transcriptase is a polymerase DNA that can catalyze the polymerization of cDNA, starting with a single-strand RNA or DNA molecule. Utilization Reaction buffer This does not differ much between one supplier and another: only the relative concentrations of the components are different. Concentration Variable according to the quality of the enzyme. Temperature Optimal polymerization takes place at a temperature of between 37 and 42C. 74
Supplied with the enzyme (10X) For example, TrisHCl, pH 8.3, 400 mM KCl, 80 mM MgCl2, 10 mM DTT From 1 to 20 U/l Generally 40C recommended temperature:
4.3 4.3.3.2 M-MLV reverse transcriptase
The Tools
Origin M-MLV reverse transcriptase is a recombinant enzyme. It is cloned from an Escherichia coli strain that expresses the gene of the leukemia virus in the Moloney mouse. Its molecular weight is 71 kDa. It is an RNA- and DNAdependent DNA polymerase. It has been genetically modied so as to eliminate its RNase H activity. Quantity supplied The quantity supplied corresponds to a degree of activity of between 10,000 and 50,000 U. The conservation buffer contains 50% glycerol to avoid freezing. Conservation In order for the enzyme to retain its maximum level of activity, it should in general be stored in aliquots at 20C. Principle of action M-MLV reverse transcriptase is a DNA polymerase that can catalyze the polymerization of cDNA, starting with a single-strand RNA or DNA molecule. Utilization Reaction buffer This does not differ much between one supplier and another: only the relative concentrations of the components are different. Concentration Variable according to the quality of the enzyme. Temperature Optimal polymerization takes place at a temperature of 37C. 4.3.3.3 Tth DNA polymerase
This modication makes it possible to obtain larger fragments when cDNA is being synthesized. A unit (U) is dened as the quantity of enzyme that, in 10 min at 37C, incorporates 1 nmole of insoluble dTTP in an acid medium, starting with an RNA [poly (A)] matrix and an oligo(dT) primer. To avoid successive warmings, an isothermal refrigerated box should be used. This reaction requires a primer and Mg ions as cofactors.
2+
Supplied with the enzyme (5X) For example, 250 mM TrisHCl, pH 8.3, 500 mM KCl, 10 mM MgCl2, 40 mM DTT In general, 50 U/l
Origin Tth DNA polymerase is a recombinant enzyme. It is cloned from the bacterial Thermus thermophilus KTP strain. Its molecular weight is 92 kDa, and it is capable, in the presence of MnCl2 and at a high temperature (74C), of polymerizing DNA from an RNA matrix. Tth DNA polymerase can also polymerize double-strand DNA nucleotides from a DNA matrix in the presence of MgCl2.
Its half-life is 40 min at 95C. This high-temperature reverse transcription activity minimizes the kind of problem that can be caused by the existence of secondary RNA structures, which are unstable at high temperatures. It has been shown to be effective in synthesizing fragments of up to 12 kb.
75
Reverse Transcription (RT) Quantity supplied The quantity supplied corresponds to a level of activity varying between 100 and 1000 U. The conservation buffer contains 50% glycerol to avoid freezing. Conservation In order for the enzyme to retain its maximum level of activity, it should in general be stored in aliquots at 20C. Principle of action Tth DNA polymerase has reverse transcriptase activity in the presence of MnCl2, and DNA polymerase activity in the presence of MgCl2. Its synthesis rate is 60 nucleotides per second per enzyme molecule. Utilization Reverse transcription buffer
A unit (U) is dened as the quantity of enzyme that, in 30 min at 74C, incorporates 10 nmol of insoluble dNTPs in an acid medium. To avoid successive warmings, an isothermal refrigerated box should be used. The same enzyme can thus be used for both reverse transcription and amplication by changing the composition of the reactive mixture between the two steps.
Amplication buffer
Supplied with the enzyme (10X) For example, 670 mM TrisHCl, pH 8.8, 166 mM (NH4)2SO4, 25 mM MnCl2 The MnCl2 solution is supplied separately so that its concentration can be optimized. Supplied with the enzyme (5X) For example, 335 mM TrisHCl, pH 8.8, 83 mM (NH4)2SO4, 50 mM MgCl2, 0.1 mM EDTA, 25% glycerol, 0.1% Tween 20 The MgCl2 solution is supplied separately so that its concentration can be optimized. In general, 5 U/l The high temperature also ensures a higher degree of primer hybridization specicity and extension reaction.
Concentration Variable according to the quality of the enzyme. Temperature Polymerization is optimal at 74C.
4.3.3.4
Criteria of choice AMV M-MLV Cloned (E. coli) Moloney murine leukemia virus 10,000 to 250,000 U 20C 24 months 50 U/l 37C Mg2+ 8.3 Tth Cloned (E. coli) Thermus thermophilius 100 to 1000 U 20C 12 months 5 U/l 7274C Mn2+ 8.8
Specicity Origin
Extracted (Avian myeloblastosis virus) Quantity supplied 250 to 1000 U Storage temperature 20C or 70C Stability 24 months Concentration 20 U/l Optimal activity 4042C temperature Cofactor Mg2+ pH buffer 8.3 76
4.4
Materials/Reagents/Solutions
4.4 MATERIALS/REAGENTS/SOLUTIONS
4.4.1 Thermocycler
These are automatic, programmable machines with a heating block into which tubes or slides can be inserted. They maintain a given temperature for a given period of time. Thus, the temperature and duration of each step can be predetermined, from reverse transcription to the three steps in the PCR cycle. The necessary characteristics of the thermal cycler are: Speed Accuracy Reproducibility These qualities determine the yield and efciency of the amplication process. Different types of apparatus are commercially available, some of which are specic for liquid PCR and some for in situ PCR, while others combine the two amplication systems, using interchangeable heating blocks (tubes/ slides). Each model has both advantages and disadvantages, and rather than make comparisons between them we will simply present one of each type in an objective way.
4.4.1.1 Thermocycler for PCR, either in situ or in liquid phase This apparatus is used to carry out the reverse transcription and amplication steps on cells in suspension. The main manufacturersApplied Biosystems (Perkin-Elmer), Hybaid, MJ Research, and Biometraproduce very similar models whose heating and cooling systems are highly reliable. Applied Biosystems produces two separate systems, one for liquid phase PCR, the other for in situ PCR, while the other manufacturers have at least one model that combines the two. This apparatus is dual purpose: it can take either 24 0.2-ml tubes (A) or 16 slides (B). It can produce a homogeneous temperature (0.4C) of 0 to 100C, and temperature changes take place at 1C/s for 0.5-ml tubes, and 1.2C/s for 0.2-ml tubes. x Tube compartment, which can be replaced by a slide compartment y Control and programming screen
77
Reverse Transcription (RT) z Slide compartment
Figure 4.7 Dual-purpose thermal cycler for liquid phase PCR (A) and in situ PCR (B) (Peltier Thermal Cycler for in situ, PTC-200/ 225, MJ Research). 4.4.1.2 Thermocyclers for in situ PCR
These machines process tissue samples and cells on slides. The two models shown here operate in different ways. 4.4.1.2.1
VERTICALLY
APPARATUS
WITH
SLIDES
PLACED
This thermal cycler can take 10 slides 1.2 0.02 mm thick, placed vertically. It is equipped with the cover disk/cover clip system (see Section 4.4.2.1). It can produce a range of temperatures from 4 to 100C, and temperature changes take place at 0.67C/s. x On/Off y Slide compartment, which can hold up to 10 slides z Blocking bar { Control and programming screen Figure 4.8 Thermal cycler for in situ PCR (GeneAmp In Situ PCR system 1000, Perkin Applied Biosystems).
4.4.1.2.2
APPARATUS
WITH
SLIDES
PLACED
HORIZONTALLY
This apparatus can take 20 slides 1 mm thick, placed horizontally. It is equipped with the Easyseal system (see Section 4.4.2.2). It can produce a range of temperatures from 1.5 to 99.9C, and changes of temperature take place at 0.1 to 0.5C/s. x Control and programming screen y Compartment with a capacity of 20 slides
Figure 4.9 Thermal cycler for in situ PCR (Omnislide System, Hybaid). 78
4.4
Materials/Reagents/Solutions
4.4.2 Sealing Systems

To make sure that the reverse transcription and amplication mixtures remain in contact with the section, and that evaporation is avoided during the high-temperature cycles, an incubation chamber needs to be made on the section. The most basic way of doing this is to place a cover clip on a section coated with the reactive medium, and to seal it with a silicone of the rubber cement type. Two systems are commercially available. One of these is specic to Perkin-Elmer Applied Biosystems machines, whereas the other is suitable for all the remaining types of equipment. 4.4.2.1 The Cover disk/Cover clip system
Side view
This system is suitable for the type of PCR apparatus in which the slides are placed horizontally. However, the silicone softens at the high temperatures at which the PCR takes place, and this reduces the effectiveness of the seal. The Cover disk/Cover clip system The Easyseal system
x Tissue section on a slide y Reactive medium
z Cover disk made of soft plastic { Cover clip that seals the cover disk over the slide Figure 4.10 The system that is needed for the Perkin Biosystems apparatus, where the slides incubate vertically.
3 2 1
4.4.2.2
The Easyseal system
This is a system that works for all types of slide, and is eminently suitable for equipment in which the slides incubate horizontally. It forms a sealed chamber around the section.
3
x Tissue section on a slide y Plastic frame whose size is determined by that of the section; its adhesive lower side sticks on the slide, its upper side on the plastic cover slide z Plastic cover slide
Figure 4.11 The Easyseal system.
79
4.4.3 Reagents/Solutions
Primer See Section 4.5.3.1. Whatever the type of primer used, the storage concentration is around 10 M. Store at 20C. This is supplied separately, or in the form of a mix at a concentration of 100 mM. Store at 20C. It is generally supplied with the enzyme at a concentration of 1 mM. Store at 20C. The quality of the water is very important. It must be treated with DEPC (see Appendix B1.2); otherwise, it is very convenient to use 2 ml ampoules of sterile water. See Section 4.5.3.3. Activity can vary between 5 and 400 U/l, according to the type of enzyme used. It is a good idea to check the characteristics of the enzyme, and the protocol suggested by the supplier. Store at 20C. This is a 50-kDa protein that inhibits ribonucleases. This effect is inhibited at temperatures above 50C. The specic activity is given by the supplier (average: 40 U/l). Its composition is optimized by the supplier. It comes in 5 or 10 form. It has a high 2+ MgCl2 concentration, and indeed Mg inuences the activity of the enzyme: an excess reduces its usual reaction, whereas a shortage reduces its reactive yield. For this reason, the MgCl2 solution is sometimes supplied separately, and particularly in the case of Tth DNA polymerase. Store at 20C. The MgCl2 concentration needs to be optimized. Use only if Tth DNA polymerase is used.
Deoxynucleotide triphosphates (dNTPs)
Dithiothreitol (DTT)
Sterile water
Enzyme: reverse transcriptase
Ribonuclease inhibitor (RNasin )
Buffer (specic to the enzyme)
MgCl2 solution MnCl2 solution
4.5 PROTOCOL
The reverse transcription step is carried out after the particular pretreatments needed by each sample: See Chapter 4.
80
4.5
Protocol
Frozen tissue or parafn-embedded sections Cytocentrifuged cells, or cells cultivated on slides Cells in suspension, whether from cultures, biological uid, or even the enzymatic dissociation of tissue
The wearing of gloves and RNase free conditions are obligatory. The quantity and quality of the RNA matrix are decisive for the yield (i.e., the quantity of DNA nally obtained).
4.5.1 Reactive Medium

The mixture is prepared in sterile 0.2 ml microtubes specially designed for PCR. It contains: Reverse transcription buffer DTT dNTPs in equimolar concentration Ribonuclease inhibitor, RNasin Anti-sense, poly (T), or hexamer primer Sterile water Reverse transcriptase: AMV M-MLV Tth MgCl2 solution Final concentration: 1X Final concentration: 10 mM Final concentration: 0.5 mM Final concentration: 1 U/ l M Final concentration: 1 To a nal volume of 100 l Average nal concentration: 10 U/ l A recommendation on the number of effective units per reaction is generally given by the supplier. If this is not included in the RT buffer, it is supplied separately at a given concentration. It is then necessary to test different concentrations of between 0.5 and 4 mM, according to the suppliers recommendations. Attention: Tth DNA polymerase displays reverse transcriptase activity only in the pres2+ ence of Mn ions. Final concentration: 1 mM
MnCl2 solution
4.5.2 Reverse Transcription

4.5.2.1 4.5.2.1.1
CHAMBER
Tissue sections or cells on slides MAKING

A HERMETIC INCUBATION
An indispensable procedure
The Cover disk/cover clip system
81
Reverse Transcription (RT) x Cover disk y Cover clip with grips facing forward and fully open z Magnetic slot that allows the cover disk/ cover clip assembly to be held in position { Red indicator light that shows that the heating block is in operation | Green indicator light that shows that the temperature has reached 70C (the heating system is not used for the reverse transcription step, but to carry out a hot start before the amplication step; see Section 4.5.2) } Heating platform on which the slide is placed; after the cover clip has been put in place, the slide can move to the left between two runners ~ Blocking system Compression arm knob Figure 4.12 Sealing system assembly tool (PE Applied Biosystems): open position. ~ Compression arm knob Blocking system in the closed position Sliding handles (in the direction of the arrows); this action pushes the sliding grips of the Ampli Cover Clip under the slide, anchoring the Ampli Cover Disk to the slide.
Figure 4.13 Sealing system assembly tool (PE Applied Biosystems): closed position. a. Place the slide on the platform with the rst section facing the alignment marks. The apparatus must not be switched on at this stage, as a temperature of 70C would inhibit the activity of the enzyme.
Figure 4.14 Putting the slide in position. b. Place the cover disk in the cover clip, and attach the combination to the magnetic slot on the compression arm. c. Place 20 to 30 l of the reaction mixture on the sections or cells. d. Seal. 82
4.5 x Front view
Protocol
y Side view, with the back of the slide uppermost
Figure 4.15 Sealed incubation chambers. e. Incubate in the thermal cycler. x Bar in loading position, which means that the slides can be inserted easily. This type of apparatus can hold ten slides.
Figure 4.16 Arrangement of the slides in the thermal cycler (PE Applied Biosystems). x Back of the slide against the heating block y, z Clips in the closed position { Heating block | Cover clip } Cover disk ~ Incubation chamber Metal strip retaining the slide against the heating block Figure 4.17 Side view of the slide in its compartment.
4 5 2 6 1 7 8
The Easyseal system
For all the other types of apparatus in which incubation takes place in a horizontal position.
a. Wipe the slide as carefully as possible around the section so that the adhesive frame sticks rmly to the slide. The adhesive frame (double-sided adhesive) exists in several sizes, and the choice will depend on the size of the section.
Figure 4.18 Placing the adhesive frame on the slide. 83
Reverse Transcription (RT) b. Remove the paper top liner from the frame.
Figure 4.19 Removal of the paper top liner. c. Place 30 to 50 l of the reaction mixture on the section. The adhesive frame marks out the edges of the incubation chamber.
Figure 4.20 Placing the reverse transcription medium on the section. d. Carefully lower the polyester cover over the frame starting at the end, where the reagent has been pipetted. This plastic cover sticks to the adhesive frame. A regular movement in the direction of the arrow. This procedure avoids the formation of bubbles.
Figure 4.21 Sealing the plastic cover. e. Incubate in the thermal cycler. x Filling the two trays that make up the damp chamber with sterile water
y Placing the slides in the holder, which can take up to 20 slides z Slide-blocking system
84
4.5
Protocol
Locking down the cover before switching on the machine, which will already have been programmed
Figure 4.22 Placing the slides in the Hybaid Omnislide apparatus. 4.5.2.1.2 INCUBATION TEMPERATURE This does not depend on the choice of primer. With random primers, however, i.e., hexamers or nonamers, incubation must be carried out for 10 min at 25C prior to incubation at the temperature that works best for the enzyme. It is advisable, in all cases, to use the temperature suggested by the manufacturer of the enzyme. The temperature that most commonly produces optimal activity is 40C. For some enzymes, this temperature can be as high as 42C. The advantage with this temperature is that it is generally the same as the hybridization temperature of the primer.
This temperature varies according to the choice of enzyme: For AMV reverse transcriptase, a temperature of between 37 and 42C is recommended. The temperature for the optimal activity of M-MLV reverse transcriptase is 37C. The temperature for the optimal activity of Tth DNA polymerase is 74C.
4.5.2.1.3 DURATION OF INCUBATION This is constant, whatever the choice of enzyme.
1h
4.5.2.1.4 DEACTIVATION OF THE ENZYME The simplest method is thermal 2 min deactivation. 94C 4.5.2.2 Cells in suspension
This is the temperature necessary for the destruction of the enzyme.
The cells are pretreated and centrifuged. The 6 concentration of the cell pellet is 2 10 cells/ml. a. Prepare the reaction medium in a sterile microtube, without adding the amount of sterile water necessary to the nal dilution. b. Place the cell pellet in suspension in the calculated amount of sterile water. c. Add the reactive medium, and homogenize by careful pipetting. d. Add the reverse transcriptase. 200 U/l e. Homogenize further by careful pipetting. f. Carry out the incubation in a liquid-phase PCR apparatus.
See Chapter 3. See Section 4.5.2.
85
Reverse Transcription (RT) 4.5.2.2.1 INCUBATION TEMPERATURE The incubation temperature depends on the enzyme, and not the primer. 4.5.2.2.2 DURATION OF INCUBATION It is constant, whatever the choice of enzyme.
See Section 4.5.2.1.2.
1h
4.5.2.2.3 DEACTIVATION OF THE ENZYME a. Add a heating step to the RT 2 min procedure. 94C b. Lower the temperature. 4C
This temperature is necessary for the destruction of the enzyme. This temperature allows the slides to be left in the PCR apparatus without risk.
4.5.3 Washing
4.5.3.1 Tissue sections or cells on slides This is a delicate step, during which the tissue section or cells risk being damaged due to a suction effect. The removal should be carried out as gently as possible. This step is carried out in a tray. This prevents the phosphate from precipitating in the alcohol baths and producing white streaks.
a. Remove the combination of cover disk and cover clip, the Easyseal system, or simply the sealed cover clip, as the case may be. b. Wash in a 0.1 M sterile phosphate buffer. c. Rinse in sterile 9 NaCl. 5 min 2 min
d. Dehydrate in alcohol baths 2 min of increasing concentration: per bath 95, 100. e. Allow the slides to dry under the hood. Following step Amplication by PCR 4.5.3.2 Cells in suspension
The slides can be stored in a box with a desiccant at room temperature (for rapid utilization) or 20C (for longer conservation). See Chapter 5.
a. Centrifuge.
2 min 1500 g b. Eliminate all the supernatant, and put the cells back in suspension in about 200 l of PBS. c. Centrifuge. 2 min 1500 g d. Eliminate the supernatant, and put the cells back in suspension in 100 l of PBS. Following step Amplication by PCR
In most cases, the cell pellet is clearly visible.
The cells are then directly usable for PCR. They can, however, be frozen and stored at 80C in aliquots of 10 or 20 l. See Chapter 5.
86
Chapter 5
Contents
CONTENTS
5.1 Principles of In Situ PCR and RT-PCR . . . . . . . . . . . . . . . . . . . . . . 5.1.1 5.1.2 5.1.3 5.2 5.3 Amplication of Double-Stranded DNA . . . . . . . . . . . . . . . Amplication of Reverse-Transcribed cDNA . . . . . . . . . . . Exponential Amplication . . . . . . . . . . . . . . . . . . . . . . . . . . 91 92 93 94 95 96 96 96 96 96 96 99 99 100 100 100 101 101 101 102 102 103 104 104 104 105 107 107 108 108 109 109 109 110 110 110 111
Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tools. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.1 Deoxynucleotide Triphosphates (dNTP) . . . . . . . . . . . . . . . 5.3.1.1 Characteristics. . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.1.2 Working Concentration. . . . . . . . . . . . . . . . . . . . . 5.3.1.3 Labeled Deoxynucleotides . . . . . . . . . . . . . . . . . . 5.3.1.3.1 Antigenic Labels . . . . . . . . . . . . . . . . . 35 33 5.3.1.3.2 S and P Radioactive Labels . . . . . . 5.3.2 Primers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.2.1 Characteristics. . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.2.2 Position on the Gene Structure . . . . . . . . . . . . . . . 5.3.2.3 Primer Hybridization Temperature . . . . . . . . . . . . 5.3.2.4 Conservation and Storage . . . . . . . . . . . . . . . . . . . 5.3.2.5 Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.2.6 Validation of the Primer Pair by Liquid-Phase PCR. . . . . . . . . . . . . . . . . . . . . . . . . 5.3.2.7 Labeled Primers . . . . . . . . . . . . . . . . . . . . . . . . . . 35 33 5.3.2.7.1 Radioactive Labels ( S or P) . . . . . . 5.3.2.7.2 Advantages/Disadvantages . . . . . . . . . 5.3.2.7.3 Nonradioactive Labels. . . . . . . . . . . . . 5.3.2.7.4 Advantages/Disadvantages . . . . . . . . . 5.3.3 Enzymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.3.1 Taq DNA Polymerase . . . . . . . . . . . . . . . . . . . . . . 5.3.3.2 Other Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.3.2.1 Pfu and Tgo DNA Polymerase . . . . 5.3.3.2.2 Extrapol DNA Polymerase . . . . . . . . 5.3.3.2.3 Tth DNA Polymerase . . . . . . . . . . . . . 5.3.3.3 Criteria of Choice . . . . . . . . . . . . . . . . . . . . . . . . .
5.4
Equipment/Reagents/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.1 5.4.2 Thermocyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sealing Equipment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.2.1 The Different Systems . . . . . . . . . . . . . . . . . . . . . 5.4.2.2 Sealing Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . Reagents/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4.3
89
Polymerase Chain Reaction (PCR) 5.5 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.1 Reaction Mixture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.1.1 Direct PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.1.2 Indirect PCR/RT-PCR . . . . . . . . . . . . . . . . . . . . . . The Hot Start . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.2.1 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.2.2 Avoiding Hot Starts. . . . . . . . . . . . . . . . . . . . . . . . The Amplication Cycles . . . . . . . . . . . . . . . . . . . . . . . . . . Number of Cycles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Programming the Thermocycler . . . . . . . . . . . . . . . . . . . . . The Particular Case of Cells in Suspension . . . . . . . . . . . . . Washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.7.1 Washing and Postxation of Sections or Cells on Slides . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.7.2 Washing Cells in Suspension . . . . . . . . . . . . . . . . 112 112 112 113 114 114 114 115 115 116 117 117 117 118
5.5.2
5.5.3 5.5.4 5.5.5 5.5.6 5.5.7
90
5.1
Principles of In Situ PCR and RT-PCR
Since 1985, genic amplication techniques of the PCR (polymerization chain reaction) type have made it possible to exponentially amplify, in vitro, specic nucleotide sequences of which there may be as little as a single copy. PCR and RT-PCR can thus be used to neosynthesize a large number of copies of a specic DNA or RNA sequence. In situ PCR and RT-PCR combine the advantages of genic amplication with the intracellular localization of nucleotide sequences visualized by in situ hybridization. It is thus a highly sensitive method, which can be used with parafnembedded sections, frozen-tissue sections, cells that have been centrifuged or cultured on slides, or cells in suspension.
Gloves must be worn. RNase-free conditions are very important. All the equipment must be sterilized at 180C for 3 h, and water used for solutions must be sterile or DEPC-treated (see Appendix B1.2).
5.1 PRINCIPLES OF IN SITU PCR AND RT-PCR

Sequences of interest are amplied within cells in the presence of: Specic short primers (synthetic oligonucleotides) which ank the target sequence dNTPs A thermostable enzyme, e.g., Taq DNA polymerase In a reaction environment (buffer, MgCl2, etc.) An amplication cycle comprises three key steps: Denaturation of the target sequences See Chapter 1. This step is accomplished by breaking hydrogen bonds at high temperatures (94 to 100C). This step has to be optimized according to the particular characteristics of the primers. The hybridization temperature can be anywhere between 50 and 70C. This is due to the DNA polymerase activity of the enzyme and the dNTPs added to the reaction medium. The number of cycles has to be optimized according to the nature of the tissue and the target sequence. 91 See Section 5.3.2. See Section 5.3.1. See Section 5.3.3.
Hybridization of the primers
Extension of the primers by copying the DNA template Each cycle is repeated 20 to 30 times.
5.1.1 Amplication of Double-Stranded DNA

1st cycle
Endogenous DNA
3'
5' 1
5'
3'
x Denaturation at 94C to separate out and linearize the two DNA strands
3' 5'
5' 3' 2 5' 5' 3' 3 5' 3' 5' 4 3' 5' 5' 5' 3' 5' 3' 3' 5'
3' 5'
y Hybridization of primers on the complementary sequence of each DNA strand; this step is generally carried out at a temperature of 5060C
3' 5'
z Extension: Taq DNA polymerase attaches to the 3 end, and adds the dNTP present in the medium, in the 3 5 direction, using the complementary strand as a model. Neosynthesis begins when the optimal reaction temperature of the enzyme is reached (i.e., 7274C, depending on the manufacturer).
3'
{ By the end of the rst cycle, two double strands of DNA have been obtained.
3'
5'
2nd cycle 5' 3' 5' 3' 2 3 5' 3' 5' 3'
x Denaturation
y Hybridization z Extension
92
5.1
5' 5' 3' 5' 5' 5' 5' 5' 3' 4 5' 3' 3'
Principles of In Situ PCR and RT-PCR
{ By the end of the second cycle, four double strands of DNA have been obtained. The amplication is exponential.
5' 3' 5' 3'
5' 3' 5' 3'
By the end of the second cycle, two copies of the sequence of interest have been obtained.
3'
5' 3' 3' 5'
5' 5'
Figure 5.1 The PCR principle, the rst and second amplication cycles.
5.1.2 Amplication of Reverse-Transcribed cDNA

1st cycle 3' 3' 1 5' 3' 2 3' 5' 3' 3 3' 5' 3' 4 5' 3' 5' 5' 5' 3' 5' 5'
RNA cDNA obtained by reverse transcription from an anti-sense primer x Denaturation at 94C: the cDNA strand separates out from the RNA strand and linearizes y Hybridization of the sense primer on the complementary cDNA sequence at 5060C z Extension by Taq DNA polymerase, which can attach only to the 3 end of a DNA sequence { It is thus only at the end of the rst cycle that the double-stranded form reappears (see Figure 6.1). After the denaturation that takes place during the second cycle, the two primers can position themselves, and the sequence of interest will be synthesized in the same way by the end of this cycle. Figure 5.2 PCR, using cDNA obtained by reverse transcription of the target RNA.
3'
93
5.1.3 Exponential Amplication

With each cycle the number of DNA strands doubles, so that the repetition of the cycles leads to the exponential amplication of a specic nucleotide sequence according to the theoretical formula: n number of copies = 2 where n represents the number of cycles.
3 5 3 5 3 5
In other words, 20 amplication cycles pro20 duce 2 copies. The yield is never 100%; for liquid-phase PCR it is closer to 70%. It is difcult to dene in situ. Target DNA, with the sequence of interest
5 3 5 3 3 5 5 3 5 3 3 5 3 5 3 5 3 5 3 5 3 5
3 5
First cycle: No fragment of the desired size has been synthesized. Second cycle: By the end of this cycle, two fragments of the required size have been obtained.
5
5 3
3 3 5 3 5 3 5
Third cycle: Two double strands of the required size have now been synthesized.
5
Fourth cycle: Eight double strands have now been synthesized. Figure 5.3 Exponential amplication of a sequence of interest during the rst four PCR cycles.
94
5.2
Diagram of the Different Steps
Paraffin sections
Frozen sections Cell smears
Cell suspensions
Pretreatments
RNA
DNA
Reverse transcription
Amplification
Washing
Washing
Amplification
Fixation of amplified product
Washing
95
5.3 TOOLS
5.3.1 Deoxynucleotide Triphosphates (dNTP)
5.3.1.1 Characteristics Their degree of purity, as given by the manufacturers, is generally >98%. In liquid form, dNTPs are supplied at a concentration of 100 mM.
These are generally supplied either: In liquid form, at pH 7, in ultrapure water or in lyophilized form or Separately (dATP, dCTP, dGTP, dTTP), or in equimolar mixtures 5.3.1.2 Working concentration An excess of dNTP can bring about the amplication of nucleotides that have been poorly, or not at all, incorporated, thus reducing the delity of the enzyme.
The usual concentration is between 50 and 250 M; an equimolar concentration ensures the delity of the enzyme.
5.3.1.3
Labeled deoxynucleotides For direct in situ PCR
Numerous authors have advised against the direct incorporation of labeled nucleotides during the amplication step. The number of nonspecic incorporations, and thus false positive results, can be signicant. These nucleotides are nonetheless used to label primers. 5.3.1.3.1 ANTIGENIC LABELS These are nucleotide triphosphates with nitrogenous bases onto which antigenic molecules have been grafted by chemical coupling. The most commonly used reagents are biotin, digoxigenin, and uorescein. Biotin Also known as vitamin H, its molar mass is 244, and it has a particular afnity for:
These labels are grafted by substitution of a thymidine CH3 radical, which turns them into deoxyuracil (dUTP). Afnity: 10 M Note: Many types of tissue, such as the liver, the intestine, and the endometrium have large amounts of endogenous biotin, which can cause interference during the detection of biotinylated amplied products. Glycoprotein extracted from egg white Glycoprotein extracted from a Streptomyces avidii culture medium
15 1
Avidin (or extra-avidin) Streptavidin
96
5.3 It can also be detected by an anti-biotin antibody. Labels: These proteins, and the antibody, can be conjugated with: An enzyme or A uorochrome or Colloidal gold
O O C O O- P O P O P O O HN 3 4 5 O N H H N O O N H HN H S NH H
Tools
Polyclonal or monoclonal
Peroxidase or alkaline phosphatase Fluorescein, Rhodamine, Cyanine 3, Alexa, etc. Visualization of the amplied product by electron microscopy; see Section 8.13. The biotin molecule X-dUTP biotin, where X represents the number of carbon atoms (between 6 and 21) that separate the biotin molecule from the base The most commonly used labeled nucleotide is 11-dUTP biotin. Figure 5.4 A biotinylated nucleotide.
OH OH OH CH 2 5
C 2 1 6 CH O N 1 2 R
4 3 OH
Uracil nucleotide
Attached carbon chain
biotine
Advantages Four xation sites for avidin or streptavidin Biotin-labeled nucleotides can be incorporated directly during the amplication process Low molecular masss Disadvantages Its utilization is limited by the fact that many types of tissue contain endogenous biotin. The specicity of anti-biotin sera must be checked. Digoxigenin Extracted from digitalis (Digitalis purpurea or D. lanata). Its molar mass is 300 to 400. Revelation by an anti-digoxigenin IgG, which can be conjugated to: An enzyme A uorochrome Colloidal gold The use of blocking agents (see Appendix B6.2.1) Inhibition by preincubation with a solution of biotin (homologous antigen) An antigenic molecule, which does not exist in animal tissue Either polyclonal or monoclonal Alkaline phosphatase or peroxidase Fluorescein, Rhodamine, Cyanine 3, Alexa, etc. Visualization of the amplied product by electron microscopy (see Section 8.13) The efciency of the detection process Notably for the detection of viral DNA, e.g., the human papilloma virus (HPV) (see Chapter 11; Figures 11.1 to 11.3) Small steric hindrance, which favors the incorporation of the labeled nucleotide
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OH O
The digoxigenin molecule
CH 3 OH CH3 O HN C 4 H O N (CH2)5 N H O O H N N N O
O O O O P P P O
OH OH OH CH 2 5
O C 2 1 6 CH O N 4 1 3 2 OH R
O O
X-dUTP digoxigenin, where X represents the number of carbon atoms (between 6 and 21) that separate the biotin molecule from the base The most commonly used labeled nucleotide is 11-dUTP digoxigenin.
Uracil nucleotide
Figure 5.5 Nucleotide coupled to digoxigenin.
Advantage Detection is specic to the animal kingdom. Disadvantages Structure of digoxigenin similar to that of steroids Toxicity A high molecular masss Fluorescein This is a uorochrome which, when stimulated by a photon, emits another photon of higher wavelength. This property is responsible for the uorescence produced by ultraviolet radiation. Nonspecic bonds can be formed with some receptors. This must not be inhaled. This limits its incorporation. Mw: 332 Not its most useful property; this compound is generally considered an antigen that can be detected by an antiuorescein antibody. Animal tissue does not contain any endogenous digoxigenin.
OH
The uorescein molecule

C O C HN 4 3 5 2 O HO O+
O O O
6 CH O- P P P O O C 1 N O OH OH OH CH2 1 4 5 3 2 OH R
X-dUTP uorescein, where X represents the number of carbon atoms (generally 11 or 16) that separate the uorescein molecule from the base
Uracil nucleotide
Figure 5.6 Nucleotide coupled with uorescein.
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5.3 This labeled nucleotide can be incorporated directly during the amplication process, but it is more generally used to label primers during their synthesis. Fluorescein, like other uorochromes, is especially used in PCR and RT-PCR techniques with cells in suspension. Advantages The possibility of visualizing the amplied product directly A rapid method The possibility of amplifying the signal by an immunohistochemical reaction Disadvantages The disadvantages generally associated with uorochromes Specic equipment required for visualization Fluorescence poorly conserved over time
Tools
In both cases, the amplication product can be visualized directly by uorescence microscopy. Its detection by an immunocytochemical reaction amplies the signal. Flow cytometry makes it possible to detect rare positive cells.
This is possible providing the target sequence is sufciently strongly expressed. It allows different pairs of primers to be tested rapidly. This takes longer, but is also more sensitive.
Despite storage of the slides at 4C, and the antifading substance that is added to current embedding media
Low sensitivity in direct visualization 5.3.1.3.2 S AND P RADIOACTIVE LABELS Radioactive nucleotides are not used in direct incorporation, but may be used to label directPCR primers.
35 33
As well as a lack of specicity, there is a high risk of contamination. See Section 5.3.2.7.
5.3.2 Primers
The primers are a pair of synthetic oligonucleotides whose sequence is complementary and anti-sense to each of the DNA strands. Although the particular characteristics of each primer are identical to those of the specic anti-sense primer used for the reverse transcription, there are some rules that have to be respected regarding their position on the structure of the gene. A pair of primers is generally enough to produce amplication, although another pair of primers situated on the sequence amplied by the rst pair of primers may also be used. cDNA produced by reverse transcription is double-stranded after the rst PCR cycle. See Section 4.3.1.
This method is known as nested PCR. It makes possible a further amplication of the product after its amplication by the rst primers, thus increasing sensitivity and specicity.
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Polymerase Chain Reaction (PCR) 5.3.2.1 Characteristics The degree of homology with other sequences that might be expressed in the tissue under consideration has to be checked against data banks. False-positive results are usually caused by primer mismatches (see Section 9.4). Autoligation phenomena Palindromic sequences
A primer consists of a sequence of 20 to 22 mers, containing 50 to 55% GC, which is anti-sense and complementary to a single sequence of the target DNA. Sequences that could give rise to secondary structures should be avoided. The 3 ends of the primers must not be complementary if the formation of dimer is to be avoided. 5.3.2.2 Position on the gene structure
The primers must enclose the sequence of interest. In situ PCR necessitates a pair of primers capable of generating fragments >400 bp, although PCR efciency is higher, and especially in the liquid phase, if primers that generate 150 to 300 bp fragments are used. To avoid the amplication of genomic DNA, it is best to choose sequences from different exons that enclose one or more introns.
Each 3 end terminates with a primer. Purpose is to limit problems of diffusion. Smaller fragments could diffuse through the nuclear and cytoplasmic membranes, even after permeabilization during the pretreatment steps. If the primers that enclose the sequence of interest in the cDNA also enclose one or more introns in the genomic DNA, the sequence to be amplied will be much too long, and the extension phase too short, to allow this synthesis to take place. cDNA is made up of the exons as a whole. I = Introns E = Exons Sense and anti-sense Figure 5.7 The positions of the primers on the structure of the gene.
A gene is dened by its exons, which make up the totality of the protein-coding region.
ATG 5 TATAA E1 5 E2 3 E3 E4 3 E5 5 I1 I2 I3 I4 I4 3
5.3.2.3
Primer hybridization temperature The ideal situation is when the two primers hybridize at the same temperature. Tm = melting temperature. A higher hybridization temperature would not be a problem: the smaller the difference between the hybridization temperature and the extension temperature, the better the thermocycler performs.
The hybridization temperature will be that of the primer with the lowest Tm. This is generally around 54C
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5.3 Example of a calculation for a 22-mer oligonucleotide with 60% GC in 50 mM KCl: Tm = 81.5 + 16.6 (log[KCl]) + 0.41 (% GC) 675/22 Tm = 81.5 + 16.6 (log[0.05]) + 0.41 (60) 675/22 Tm = 81.5 + 16.6 (1.30) + 24.60 30.68 Tm = 53.84C 5.3.2.4 Conservation and storage
Tools
According to Breslauers formula (see Section 3.1.3).
Oligonucleotides are generally supplied in lyophilized form. They are reconstituted in DEPC-treated water. Storage is at 20C. 5.3.2.5 Concentration
This guarantees their stability during transport, and allows the user to reconstitute them at the chosen concentration (e.g., 10 M). Or in TE buffer.
Primers must be at an equimolar concentration in the reaction mixture. This concentration will need to be optimized.
But the addition of both primers at a concentration of 50 pmoles is a good starting point. Final concentration: 1 M, for a reaction volume of 50 l
5.3.2.6
Validation of the primer pair by liquid-phase PCR It is necessary to identify the gene of interest by liquid-phase RT-PCR in RNA extracted from the tissue being studied, prior to determining its cellular location by in situ PCR. See Appendix A4. Validation of the methods, hybridization temperatures, and products Verication of size by comparison with the molecular-weight labels Can be analyzed by sequencing of the amplied DNA, after extraction from agarose The amplied fragment does indeed correspond to the expected 427 bp fragment, as can be seen in lines 2, 3, and 5. Line 2 = PCR on the positive-control DNA Line 3 = PCR specic to the DNA being studied Line 4 = Negative PCR: No band is visible Line 5 = Nonspecic PCR: The parasite bands are due to illegitimate hybridizations between primers and DNA Figure 5.8 Analysis of amplication products on agarose gel. 101
Before performing in situ PCR or RT-PCR, the primers should be tested by liquid-phase PCR or RT-PCR on control cDNA or RNA extracted from tissue or cells known to express the gene being sought. The amplication product is analyzed by electrophoresis on agarose gel to nd out whether: Amplication has occurred The amplied product does or does not correspond to the expected fragment The amplied product does or does not correspond to the gene being studied
1 1000 bp 500 bp
427 bp
Polymerase Chain Reaction (PCR) When the amplied product has been obtained, liquid-phase PCR is also an excellent way of optimizing the amplication conditions which often reduces to looking for the optimal primer hybridization temperature. For example, raising the hybridization temperature by a few degrees may be enough to eliminate the parasite bands.
50 52 54 56 58 60C
This ideal temperature is the starting point for the adjustment of the in situ PCR.
The temperature should be that which gives the best signal without parasite bands (in this case, 56C).
427 bp
Figure 5.9 Optimization of the primer hybridization temperature. If, in spite of this rise in temperature, there are still parasite bands, it will be necessary to: Reduce the number of cycles Lower the concentration of the primers If these different modications bring about no improvement, other primers will have to be found before in situ PCR can be carried out. And thus more specic hybridization From 30 to 25 or 20 cycles Which, in excess, can hybridize in a nonspecic way In in situ PCR, if one or both primers hybridize in a nonspecic way, it is impossible to tell false positives from valid results, and the data are impossible to interpret.
5.3.2.7
Labeled primers See Section 9.4. See Chapter 11, Figure 11.1. Ease of use and rapidity The labeling of the primer must be carried out in the 5 position, or through the incorporation of labeled nucleotides during oligonucleotide synthesis. The labeling system depends on the particular label used. Labeling in the 3 position leads to the hybridization of this end of the target sequence. The positioning of the enzyme reduces the efciency of the extension, and thus the amplication. [ S]-dATP (or -dCTP) and [ P]-dATP (or -dCTP) are available from most suppliers of molecular biology products.
35 33
The use of labeled primers in a direct PCR protocol is considered as the least specic method, and is not recommended. It is, however, potentially useful, notably with uorescent labels.
5.3.2.7.1 RADIOACTIVE LABELS ( S OR P) The labeling uses the enzymatic activity of the polynucleotide kinase to insert a radioactive nucleotide at the 5 position of the primer. This nucleotide is itself labeled on its phosphate group at the position. The labeling process thus consists of phosphorylating the 5 end by adding a phosphate.
35
33
102
5.3
Tools
S or P labeling of the phosphate group in the position

35 33
The addition reaction is carried out by polynucleotide kinase (an enzyme extracted from calf thymus).
Figure 5.10 Labeling by 5 extension. Probably due to the emission, at high temperatures, of radioactive H2S Whose half-life is relatively short (25 days)
Besides the lack of specicity of the direct method, serious contamination problems have been mentioned in relation to radioactivity, and 35 in particular with S. Those who insist on using radioactivity should opt 33 for P. 5.3.2.7.2 ADVANTAGES/DISADVANTAGES
Advantage Sensitivity Disadvantages The cost, along with the specic problems inherent in the manipulation of radioactive substances 33 Low cell resolution; with P, it is around 15 to 20 m There is a need for radioprotection. This is a major disadvantage, as the aim of in situ PCR is to identify the cells that express the gene of interest within a given cell population.
Low specicity due to the use of the direct method
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Polymerase Chain Reaction (PCR) 5.3.2.7.3 NONRADIOACTIVE LABELS Antigenic molecules such as biotin, digoxigenin, alkaline phosphatase, and the uorescent molecules are either:
s
Chemically coupled to the modied 5 end of a synthetic oligonucleotide, in which case the addition of an aliphatic amine group to the 5 end makes possible the conjugation of different molecules, or Incorporated during the synthesis of the oligonucleotide. Several labeled nucleotides can thus be incorporated, and this increases the efciency of the labeling. 5.3.2.7.4 ADVANTAGES/DISADVANTAGES
This amine modication of the 5 end of the synthetic oligonucleotide is available from a number of suppliers. Several labels are also available at the 5 end, e.g., biotinylation, uorescein, rhodamine, alkaline phosphatase, peroxidase. This method is used mostly for uorescent labels. It reduces the efciency of the hybridization process.
Advantages Easy to use Rapid Two PCRs can be carried out simultaneously, using primers labeled with uorochromes of different colors. Disadvantages This method does not give the same degree of specicity as the direct method. The observation of uorescence requires a suitable microscope. It is impossible to conserve samples processed by uorescence. See Section 9.2. Confocal microscopy provides a satisfactory degree of precision. Specic equipment is not required. This option, which is used mostly with cell suspensions, makes it possible to visualize two sequences of different genes in the same cell or different cells.
5.3.3
Enzymes
It is derived from a thermophilic bacterium, Thermus aquaticus, which lives in thermal springs at 70C. All the different suppliers now offer complete ranges of enzymes, corresponding to the diversity of users needs. This is derived from Pyrococcus furiosis, which ourishes at 100C in geothermal marine sediments. It is very stable. Its level of DNA polymerase activity is very low at temperatures <50C, which avoids hot starts.
The most commonly used enzyme is Taq DNA polymerase, which can be obtained from any supplier of molecular biology products, modied to a greater or lesser extent to maximize its thermal stability, ease of use, and effectiveness. Pfu DNA polymerase, which has a different origin and has appeared on the market more recently, presents some advantages.
104
5.3 The aim, here, is not to present an exhaustive, comparative list of all currently available enzymes, but to set out their most signicantand most usefulproperties. 5.3.3.1 Taq DNA polymerase
Tools
Origin This thermostable DNA polymerase is most often recombinant. It is obtained from the recombinant bacterial strain E. coli, which expresses the YT1 gene of Thermus aquaticus. Its molecular weight varies between 85 and 94 kDa. Presentation The quantity corresponds to a level of activity of 250 to 1000 U. Its concentration is generally 5 U/l. Composition of the conservation buffer is the following: 20 mM TrisHCl, pH 8 (25C); 100 mM KCl; 0.1 mM EDTA; 1 mM DTT; 50% glycerol; 0.5% Tween 20; 0.5% Igepal. A blue or red stain is sometimes added to this buffer (e.g., Eurobluetaq or Red Taq ).
Cloning the enzyme allows a high degree of reproducibility between one batch and another. The weight depends on how it is produced. A unit is dened as the quantity of enzyme needed to transform 10 nmol dNTP into insoluble DNA in 30 min at 74C in an acid medium. The 50% glycerol prevents freezing at 20C. This eliminates the risk of forgetting about the enzyme. This is a way of checking the homogenization of the enzyme in the reaction buffer. To increase the thermal stability of their enzymes, some manufacturers carry out aminoacid substitutions in several places at the N terminal end of the protein. Concentration: 10X Concentration: 50 mM
Most enzymes are delivered at room temperature, which is possible due to their high degree of thermal stability. The enzyme is supplied with its corresponding reaction buffer; the suppliers guarantee depends on its use. The MgCl2 solution, which is a cofactor of the enzyme, is supplied separately. In this way it can be used at a range of concentrations to optimize its effectiveness. Conservation For the enzyme to remain at its maximal level of activity, it should be stored at 20C. Some manufacturers guarantee 2 years of stability in such conditions. Principle of action Taq DNA polymerase catalyzes the polymerization of nucleotides into double-strand DNA in the 5 3 direction in the presence of Mg. Some of them display 5 3 exonuclease activity. Some enzymes make possible the extension of large fragments, and the users choice will take into account, among other things, the stated effectiveness.
To avoid successive reheatings, an isothermal refrigerated box should be used.
The supplier should guarantee that the enzyme has no contaminating endonuclease activity and should state whether or not it has any exonuclease activity. Some enzymes can amplify fragments of 100 bp to 20 kb, but in most cases the enzyme should be suited to the desired extension. 105
Polymerase Chain Reaction (PCR) For in situ PCR and RT-PCR, the most important properties for an enzyme to possess are: Processivity Fidelity For in situ PCR, the size of the fragment to be amplied should be >300 bp, but never >1 kb. Taq DNA polymerases incorporate, on average, 500 bp in 15 to 20 s. The general error rate for a Taq DNA poly4 merase is of the order of 10 (i.e., one erroneous nucleotide per 10,000 incorporations). At 95C, the half-life varies between 1 and 3 h, depending on the enzyme. For example: 100 mM Tris-HCl, pH 8.8; 500 mM KCl; 1% Triton X100
Thermal stability Utilization Reaction buffer Reaction buffers are adapted and optimized so that the enzyme will be optimally effective. The components of a given buffer tend to be more or less the same from one supplier to another, but their concentrations may vary considerably. Concentration It must be optimized; 0.05 U/l is a good starting point. Cofactor The MgCl2 concentration must be empirically determined by making a range of increasing concentrations. 2+ Mg inuences the enzyme activity and the stabilization of the double strand, and raises the Tm. It also forms soluble complexes with dNTPs, which are recognized and utilized by the enzyme.
2.5 to 5 U/50 l reaction buffer Or, with some suppliers, MgSO4 0.5 to 4 mM MgCl2 is complexed mole by mole with EDTA. An excess of MgCl2 reduces the delity of the enzyme. A lack of MgCl2 reduces the reaction yield.
Final MgCl2 Concentration 1.5 mM Buffer 10 MgCl2 dNTP Enzyme Sense primer Anti-sense primer H2O Final volume 10 l 3 l 10 l 1 l 5 l 5 l 66 l 100 l 2 mM 10 l 4 l 10 l 1 l 5 l 5 l 65 l 100 l 2.5 mM 10 l 5 l 10 l 1 l 5 l 5 l 64 l 100 l 3 mM 10 l 6 l 10 l 1 l 5 l 5 l 63 l 100 l 3.5 mM 10 l 7 l 10 l 1 l 5 l 5 l 62 l 100 l 4 mM 10 l 8 l 10 l 1 l 5 l 5 l 61 l 100 l Final Concentration 1X 1.54 mM 250 M 5U 0.5 M 0.5 M
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5.3 Temperature Taq DNA polymerase attached to the 3 end of a primer synthesizes a strand complementary to each of the original strands at an optimal temperature of 72 to 74C (depending on the supplier).
Tools
The activity of the polymerase is optimal at around 74C, but it begins to appear at much lower temperatures, thus causing primer mismatching, which leads to undesired extensions and, in the end, artifactural amplication products. It was to avoid this disadvantage that the hot start technique was developed, and some manufacturers have produced quite ingenious systems. This system limits the risk of nonspecic amplications. The use of this enzyme avoids hot starts.
Inhibition of DNA polymerase activity at low temperatures By the addition of an antibody: Taq DNA polymerase is placed in the presence of an antibody, which inhibits its action up to the rst denaturation cycle, where the temperature of >90C, dissociates the enzyme/ antibody complex. When the antibody is denatured, Taq DNA polymerase activity is restored. By the use of parafn micropellets containing MgCl2: During the rst denaturation cycle, 2+ the parafn melts, thus liberating the Mg needed for the enzyme to work properly. 5.3.3.2 Other enzymes
With this system, hot starts do not occur.
Here, only the distinctive (and useful) properties of other enzymes are presented. 5.3.3.2.1 PFU
AND
TGO DNA POLYMERASE It is their origin that gives them their high level of thermal stability.
Origin Whether native or cloned, these thermally stable DNA polymerases are derived from Pyrococcus furiosis, which lives at 100C in geothermal marine environments. Characteristics Thermal stability: Pfu DNA polymerase has a half-life of 18 to 25 h at 95C.
Reduced polymerase activity below 50C, which avoids hot starts.
This extreme thermal stability means that denaturation temperatures as high as 98C are possible, with target sequences that are rich in GC. This property reduces the number of extensions resulting from nonspecic primer mismatching, which frequently occurs with Taq DNA polymerase, whose activity is high at temperatures below 50C (i.e., the average primer hybridization temperature).
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Taq DNA Polymerase Pfu DNA Polymerase
100 Polymerase activity percentage 80 60 40 20 0 15 35 50 60 70 80 Temperatures 95
The activity of the two recombinant enzymes is compared, for equal concentrations and for temperatures of 15 to 100C. Taq DNA polymerase activity increases from 17 to 70% between 35 and 50C, whereas activity of Pfu DNA polymerase increases only from 2 to 8% between 35 and 50C.
Figure 5.11 Comparison of Taq and Pfu DNA polymerase activity as a function of temperature.
(Data provided by Stratagene.) 5.3.3.2.2 EXTRAPOL DNA POLYMERASE For greater thermal stability
Origin This enzyme is obtained from the recombinant bacterial strain E. coli, which expresses the Thermus brockianus gene. Characteristics Thermal stability Extrapol has a half-life of 3 h at 96C. Fidelity Its error rate is only half that of a classical Taq DNA polymerase. 5.3.3.2.3 Tth DNA POLYMERASE
Origin This is a recombinant enzyme that is cloned from the bacterial strain Thermus thermophilus. Characteristics It catalyzes the polymerization of nucleotides in double-strand DNA, in the presence of MgCl2. It also polymerizes DNA, using an RNA template in the presence of MnCl2.
DNA polymerase activity Reverse transcription activity
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5.4 5.3.3.3 Criteria of choice Classical Taq DNA Polymerase Thermus aquaticus (cloning) 5 U/l Room temperature 20C
Equipment/Reagents/Solutions
Enzyme Origin and method of production Concentration Transport temperature Storage temperature Maximum size of the fragment to be amplied Thermal stability (halflife at 95C) Optimal operating temperature Cofactor Reaction buffer pH 5 3 exonuclease activity Processivity Fidelity (error rate) Stability
Extrapol
Pfu or Tgo DNA Polymerase
Tth DNA Polymerase Thermus thermophilus (cloned) 5 U/l Room temperature 20C
Thermus brokianus (extracted or cloned) 5 U/l Room temperature 20C
Pyrococcus furiosus (extracted or cloned) 2.55 U/l Room temperature 20C
1.8 kb
625 kb
100 bp40 kb
12 kb
1h
3h
1825 h
1h
72C Mg
2+
72C Mg
2+
72C Mg
2+
72C Mg
2+
8.8
8.8
8.75
8.8
Yes 1520 s/500 bp Classical 4 (10 ) 24 months
Yes 4045 s/kb High delity 5 (3.6 10 ) 24 months
Yes 4045 s/kb High delity 7 (4.9 10 ) 24 months
Yes 1520 s/500 bp Classical 4 (10 ) 24 months
5.4 EQUIPMENT/REAGENTS/SOLUTIONS
5.4.1 Thermocyclers
It is ease of programming and reliability that will determine the choice of apparatus. See Section 4.4.1. All the various types of apparatus give similar results, essentially differing only in terms of the number of slides they hold. 109
Polymerase Chain Reaction (PCR) The speed with which the temperatures of the different programmed stages are attained and the homogeneity of the temperature within the apparatus are also criteria for choice.
5.4.2 Sealing Equipment

For the reverse transcription step, the section must be covered with a sealed incubation chamber that will withstand the high-temperature PCR cycles. 5.4.2.1 The different systems The least sophisticated method; also the least reliable Specically designed for the Perkin-Elmer Applied Biosystems thermocycler (see Section 4.4.2.1) Suitable for all other types of thermocycler (see Section 4.4.2.2) See Section 4.4.2. 25 cycles, each lasting, on average, 4 min, at temperatures of 5595C
Place cover slips on the sections (which have been covered with the reaction medium) and seal them with a silicone of the rubber cement type. Use the cover disk/cover clip system.
Use the Easyseal system.
5.4.2.2
Sealing apparatus See Figure 4.13 and Section 4.5.2.1.1.
Apparatus is designed to carry out hot starts very easily. The heating block system over which the slide moves maintains the section and the reaction medium at a temperature of 95C, thus reducing the risk of nonspecic primer hybridization.
4 3
2 6 5 7 1
x Runners in which the slide moves as the cover disk/cover slip system is put in place y Heating block at a constant temperature of 95C (denaturation temperature) z, { Cover disk/cover slip system held in place on the magnetic plate | Red light: Heating-block indicator } Green light: Indicates that the tempera ture has attained 70C; with this system, it is easy to carry out a hot start before the amplication step ~ Blocking system Compression wheel Figure 5.12 Assembly tool sealing apparatus (PE Applied Biosystems). Open position.
110
5.4
Equipment/Reagents/Solutions
5.4.3 Reagents/Solutions
Sense and anti-sense primers See Section 5.3.2. Starting with a 100 mM solution, prepare a 10 M storage solution. Store at 20C. This is available separately, or in the form of a mixture at a concentration of 100 mM. Starting with a 100 mM solution, prepare a 10 M storage solution. Store at 20C. This is generally delivered at room temperature due to its high level of thermal stability. Store at 20C. Each enzyme has its own particular characteristics, and it is important to follow the manufacturers instructions. Its composition is optimized by the manufacturer. It is generally supplied in 10X form. Store at 20C. This is at different concentrations, and a pH of 8.3 to 9, according to the enzyme. The pH of the reaction medium has a decisive inuence on PCR efciency. The pH that gives optimum delity is 8.3; that which gives optimum sensitivity is >9. The usual compromise is somewhere around 8.8, at 25C. Ionic detergents (Tween 20, Triton 100, Nonidet P40) can be used at a concentration of 0.05% to stabilize amplication enzymes. At higher concentrations they inhibit DNA polymerase activity. If the experiment requires an excess of detergent, it is advisable to increase the enzyme concentration. Storage is at 20C. This is supplied at a concentration of 50 mM. See table in Section 5.3.3.1.
Deoxynucleotide triphosphates (dNTP)
Enzyme
A reaction buffer that is specic to the enzyme without MgCl2, or at a minimum concentration of 1.5 mM It is essentially made up of: Tris-HCl 500 mM KCl, to which may be added:
A detergent EDTA DTT
MgCl2 solution This is supplied separately in tubes, and its nal concentration in the reaction buffer needs to be optimized. DMSO (dimethylsulfoxide) If the sequences are rich in GC (>80%), secondary structures may form, which will reduce the PCR yield. Adding DMSO prevents the formation of such structures. Formamide
A concentration of 5 to 10% of the nal reaction volume. A concentration higher than 10% can totally inhibit enzyme activity.
111
Polymerase Chain Reaction (PCR) Some primer sequences may necessitate a high Tm, i.e., a temperature close to the optimal activity temperature of the enzyme (72C). This will affect the amplication. Adding formamide lowers the reaction temperature. Sterile water
Tm: melting temperature. At a concentration that has to be determined empirically. As with DMSO, a high concentration will affect the activity of the enzyme. The quality of the water is very important: DEPC water (see Appendix B1.2); 2 ml ampoules of sterile water are very practical.
5.5 PROTOCOL
The in situ PCR step can be carried out by different methods, using: Tissue sections, either frozen or embedded in parafn Cells, either cytocentrifuged or cultured on slides Cells in suspension, whether from cultures, biological uids, or even the enzymatic dissociation of tissue This amplication step is carried out either: Directly, after the pretreatment of the different samples, with the amplication of a DNA target sequence, or After the target mRNA sequence has been transformed by reverse transcription into DNA See Chapter 3. See Chapter 4. See Section 2.1.
5.5.1
5.5.1.1
Reaction Mixture
Direct PCR Gloves must be worn. RNase-free conditions are very important.
There are two ways of carrying out direct PCR: The incorporation of a labeled dNTP, or The use of labeled primers Reaction medium using a labeled dNTP In a microtube placed in ice, prepare the following mixture: PCR buffer (10X) Labeled dATP or dUTP (0.4 mM) Unlabeled dATP or dCTP (10 mM) A dNTP other than the labeled dNTP (10 mM) 112 1X 100 M 100 M 200 M
According to the manufacturer Generally biotin-14-dATP or digoxigenin11-dUTP Complementary to labeled dATP or dUTP The three other deoxynucleotides added at the same nal concentration
5.5 Sense primer (10 M) Anti-sense primer (10 M) MgCl2 (50 mM) Taq DNA polymerase (5 U/l) Sterile water 0.30.5 M 0.30.5 M 1.54 mM 12.5 U/l
Protocol
The same concentrations for the sense and anti-sense primers Necessary to optimize the nal concentration According to the enzyme Added only after 5 min of incubation at 82C, for a hot start
To a total volume of 100 l Reaction medium using labeled primers In a microtube placed in ice, prepare the following mixture: PCR buffer (10X) dNTP (10 mM ) Labeled sense primer (10 M) 1X 200 M 1 M According to the manufacturer The four deoxynucleotides added at the same nal concentration Either biotinylated, uorescent, or digoxigenin labeled Possible also to use a radioactive label (e.g., 35 33 S or P) Either biotinylated, uorescent, or digoxigenin labeled Possible also to use a radioactive label (e.g., 35 33 S or P) Necessary to optimize the nal concentration According to the enzyme Added only after 5 min of incubation at 82C, for a hot start
Labeled anti-sense primer (10 M)
1 M
MgCl2 (50 mM) Taq DNA polymerase (5 U/l) Sterile water
1.54 mM 0.10.3 U/l
To a total volume of 100 l
5.5.1.2
Indirect PCR/RT-PCR
In a microtube placed in ice, prepare the following mixture: PCR buffer (10X) dNTP (10 mM) Sense primer (10 M) Anti-sense primer (10 M) MgCl2 (50 mM) Taq DNA polymerase (5 U/l) 1X 200 M 1 M 1 M 1.54 mM 0.10.3 U/l According to the manufacturer The four deoxynucleotides added at the same nal concentration Separately or in a mixture Necessary to optimize the nal concentration According to the enzyme Added only after 5 min of incubation at 82C, for a hot start
Sterile water
To a total volume of 100 l 113
5.5.2 The Hot Start

This step is designed to avoid mismatched extension reactions. The fact is that classical Taq DNA polymerase displays a relatively high level of DNA polymerase activity at >30C. 5.5.2.1 Procedure But with the necessary quantity of sterile water, to a total volume of 100 l. This temperature can be higher, depending on the thermal stability of the enzyme. This temperature limits the number of nonspecic hybridizations. Double-strand DNA is not denatured, and a temperature of >90C is necessary. See Section 4.4.2. Or simply use sterile cover slips, carefully sealed. A considerable amount of evaporation takes place during the high-temperature cycles. It is a good idea to leave the apparatus at 82C while the reaction mixture is placed on the sections and the slides are sealed. This means that the rst cycle can start as soon as the slides are ready. It is at lower temperatures (37 to 40C) that nonspecic hybridizations occur spontaneously.
a. Prepare the reaction mixture without Taq DNA polymerase. b. Incubate the reaction mixture. 5 min 82C c. Add Taq DNA polymerase to the reaction mixture. d. Place the dry, pretreated slides on >70C the heating plate of the sealing apparatus, or a heating block. e. Immediately place 30 l of the reaction mixture on the denatured sections. f. Cover the sections with Ampli cover disks and cover clips (Perkin-Elmer Applied Biosystems), or Easylm (Hybaid). g. Place the slides in the preprogrammed thermocycler, and begin with the denaturation step.
5.5.2.2
Avoiding hot starts
A hot start is a demanding technique, but various manufacturers have developed procedures and new enzymes that allow it to be avoided, while achieving a higher quality of amplication. Blockage of Taq DNA polymerase activity by an anti-Taq DNA polymerase antibody MgCl2 embedded in parafn micropellets, as a cofactor of the enzyme
New enzymes: Pfu and Tgo DNA polymerase, whose activity is very low at <50C
It is during the denaturation step in the rst PCR cycle that the antigen/antibody complex breaks down, thus restoring the enzymatic activity of the Taq DNA polymerase. The parafn melts at around 55C, liberating the MgCl2. The enzyme is thus active only above this temperature, which is close to the specic hybridization temperature of most primers. See Section 5.3.3.2. Some of these enzymes are also coupled to an antibody to maximize the chances of obtaining satisfactory results.
114
5.5
Protocol
5.5.3 The Amplication Cycles

The PCR reaction takes place in a three-phase cycle. Denaturation phase At a temperature of 90 to 95C, the helical double strand that makes up the DNA template linearizes, and the two strands separate out. The success of this step will determine that of the entire experiment. Temperature and duration: The degree of denaturation depends 9297C on the temperature, and the length <1 min of time for which it is maintained. Hybridization phase At a well-dened temperature (Tm), the pair of primers hybridizes in a specic way, each on one of the two DNA strands, thereby specifying the part of the target sequence that will be amplied. Temperature and duration: . 5070C The hybridization temperature 3060 s depends on the nature, length, and concentration of the primers. It is 5 to 10C below the Tm. Extension phase DNA polymerase become attached to the 3 end of each primer, and synthesize the complementary strand to each of the two DNA strands. Temperature and duration: At 72C, DNA polymerases can 72C incorporate 25 to 50 nucleotides/s, which means that the duration of the 1 min extension step depends on the length of the fragment to be amplied.
If the denaturation is only partial, a return to the double-strand form will prevent the hybridization of the primers from taking place, and will lead to a false-negative result. 92C for 3060 s 97C for 1530 s See Section 5.3.2.
For a given PCR, the Tm of the two primers should be as close together as possible. It should be noted that the closer the hybridization temperature is to the optimal working temperature of the in vitro amplication enzymes, the greater the efciency of the PCR.
The optimal working temperature for DNA polymerases 15 to 30 s if the fragment to be amplied is <500 bp, 45 to 60 s if it is >1 kb
5.5.4 Number of Cycles

The amount of amplicationin other words the number of copies of a given target sequenceis directly proportional to the number of cycles. In situ amplication protocols generally comprise 20 to 30 cycles. The precise number depends on: The quantity of target DNA that is present in the tissue Number of copies = 2 , where n is the number of cycles.
n
The average number is 25, but the optimal number can only be determined empirically. For target sequences of which there are few copies, the number of cycles can be increased, although this of course entails an increased risk of nonspecic amplication. 115
Polymerase Chain Reaction (PCR) The degree of fragility of the tissue Beyond 20 cycles, the morphology of the tissue is often altered, which means that the risk of diffusion increases, with interpretational difculties as a consequence.
5.5.5 Programming the Thermocycler

The three steps that make up a PCR cycle take place at precise temperatures: Denaturation Hybridization Extension 95C 5070C 72C See Chapter 1 and Section 5.5.3. See Section 5.5.3. According to the primers used (see Section 5.5.3). See Section 5.5.3. It is the rapidity, precision, and reproducibility of the thermocycler that determine the yield and quality of the amplication step.
The programming of the thermocycler makes it possible to go from one temperature to another automatically, to maintain given temperatures for given lengths of time, and to repeat a cycle a given number of times. Denaturation Hybridization Extension 3060 s 6090 s 6090 s
According to the temperature According to the temperature According to the length of the amplied product
A time increment can be factored in for higher numbers of cycles.

100 90 80 70 60 50 40 30 20 10 0 1 3 2
x Denaturation: 30 s at 94C y Hybridization: 60 s at 55C z Extension: 60 s at 72C

2nd cycle
Temperatures (C)
1st cycle
Time (s)
Figure 5.13 PCR programming diagram.
The rst cycle begins when the temperature reaches 94C, after a phase during which the apparatus was maintained at a temperature of >80C so as to carry out a successful hot start. At the end of the last cycle it is necessary to program: A nal extension phase 72C 5 min
The time needed to prepare the slides and load them into the apparatus
116
5.5 A temperature at which the slides can be held for processing 4C
Protocol
Experience shows that after spending a certain time at 4C, sealing systems are difcult to remove, and that there is a high risk of spoiling the section. The temperature should therefore be raised (to 40 to 50C) before opening the apparatus.
5.5.6 The Particular Case of Cells in Suspension

The cells are either pretreated or washed after the reverse transcription phase. In both cases, they are in suspension in a phosphate buffer. a. Centrifuge, then remove the 2 min supernatant. 1500 g b. Add the reaction mixture to the cell pellet, and homogenize by delicate pipetting. c. Incubate the mixture. 5 min >80C d. Add the enzyme. e. Place in a thermocycler programmed for 20 to 25 amplication cycles, having checked that the tubes are rmly closed. f. Wait a nal extension phase. 5 min 72C g. Stop the reaction. 10 s 30C See Section 2.2. See Section 4.5.2.2.
For a hot start, this reaction mixture should not contain any DNA polymerase.
Final concentration: 0.1 to 0.3 U/l
The microtubes can be kept waiting for some time after the thermocycler is programmed at 4C.
5.5.7 Washing
5.5.7.1 Washing and Postxation of sections or cells on slides This is a delicate step, during which the tissue section or the cells risk damage via a suction effect. Detach as gently as possible. This step is carried out in a tray. This postxation step is necessary to the xation of the amplied products. It also stabilizes tissue structures. For tissue sections, see Appendix B4.3.2. Use for cell cultures on slides, or smears.
a. Remove the cover disk/cover slip, the Easyseal system, or the sealed coverslip. b. Wash in 0.1 M sterile phosphate buffer. c. Postx: 5 min
4% paraformaldehyde, or 70 cold alcohol d. Rinse: 0.1 M phosphate buffer 9 NaCl
1015 min 10 min 20C 5 min 2 min
See Appendix B3.4.1.
117
Polymerase Chain Reaction (PCR) e. Dehydrate in alcohol baths of 2 min increasing concentration: 95, per bath 100. f. Allow the slides to dry under a 3060 min ventilated hood. Following steps Direct observation of the amplied product
Hybridization of the amplied product with labeled probes Antigenic detection of the amplied product
After direct PCR with primers or uorescent dNTP The amount of amplication product obtained is rarely enough to permit direct visualization. Antigenic detection with an antiuorescein will then need to be considered. After indirect PCR See Chapter 6. After direct PCR See Chapter 7.
5.5.7.2
Washing cells in suspension
2 min 1500 g b. Remove all the supernatant, and resuspend in around 200 l PBS. c. Centrifuge. 2 min 1500 g d. Remove the supernatant, and resuspend in 1000 l PBS. Following steps Observation after spreading the suspension on slide, or by ow cytometry Hybridization in the case of indirect PCR with unlabeled primers Antigenic detection if the label is biotin or digoxigenin
a. Centrifuge.
See Appendix B3.4.3. The amplication product is directly visible if the amplication has been carried out in the presence of dNTP or uorescent primers. Hybridization can be carried out either in tubes or after spreading the suspension on slides (see Chapter 6). After spreading the suspension on slide (see Chapter 7).
118
Chapter 6
Hybridization
Contents
CONTENTS
6.1 6.2 6.3 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tools: The Probes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.1 6.3.2 Types of Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.2.1 Complementarity . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.2.2 Anti-sense . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.2.3 Specicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.2.3.1 Oligonucleotide Probes. . . . . . . . . . . . 6.3.2.3.2 cDNA Probes . . . . . . . . . . . . . . . . . . . Labeling the Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.3.1 Antigenic Labels . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.3.2 Radioactive Labels . . . . . . . . . . . . . . . . . . . . . . . . 35 S............................ 6.3.3.2.1 33 6.3.3.2.2 P............................ 6.3.3.3 Labeling by PCR. . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.3.3.1 Overview. . . . . . . . . . . . . . . . . . . . . . . 6.3.3.3.2 Protocol. . . . . . . . . . . . . . . . . . . . . . . . 6.3.3.3.3 Reaction Mixture for Antigenic Labeling . . . . . . . . . . . . . . . . . . . . . . . 6.3.3.3.4 PCR Protocol . . . . . . . . . . . . . . . . . . . 6.3.3.4 Labeling by 3 Extension . . . . . . . . . . . . . . . . . . . 6.3.3.4.1 Overview 6.3.3.4.2 Equipment/Reagents/Solutions. . . . . . 6.3.3.4.3 Protocol for Radioactive Probes . . . . . 6.3.3.4.4 Protocol for Antigenic Probes. . . . . . . 6.3.3.5 Purication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.3.5.1 Overview. . . . . . . . . . . . . . . . . . . . . . . 6.3.3.5.2 Equipment/Reagents/Solutions . . . . . 6.3.3.4.3 Protocol. . . . . . . . . . . . . . . . . . . . . . . . 6.3.3.6 Controls/Storage/Utilization . . . . . . . . . . . . . . . . 6.3.3.6.1 Checking the Labeling . . . . . . . . . . . . 6.3.3.6.2 Storage . . . . . . . . . . . . . . . . . . . . . . . . 6.3.3.6.3 Utilization . . . . . . . . . . . . . . . . . . . . . . 123 124 125 125 125 125 125 126 126 126 127 127 128 128 129 129 129 130 131 131 132 132 133 134 135 135 135 136 136 137 137 137 137
6.3.3
121
Hybridization 6.4 Hybridization Parameters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4.1 Hybridization Temperature . . . . . . . . . . . . . . . . . . . . . . . . . 6.4.1.1 Melting Temperature (Tm) . . . . . . . . . . . . . . . . . . 6.4.1.2 The Difference between Tm and the Hybridization Temperature . . . . . . . . . . . . . . . . . . + Na Ion Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hybridization Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Nature of the Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . Probe Length . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 138 138 138 140 140 140 140 140 140 140 140 141 141 142 143 143 143 144 144 144 144 145 145 145 145 145
6.4.2 6.4.3 6.4.4 6.4.5 6.4.6 6.5
Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5.1 Equipment/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5.1.1 Equipment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5.1.2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Reaction Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Different Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.5.2 6.5.3 6.6
Posthybridization Treatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6.1 6.6.2 Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6.2.1 Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + 6.6.2.2 Na Ion Concentration . . . . . . . . . . . . . . . . . . . . . 6.6.2.3 Washing Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.6.3 6.7
Before Revelation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.7.1 Radioactive Hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.7.1.1 Dehydration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.7.1.2 Drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antigenic Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.7.2
122
6.1
Overview
The hybridization step in the in situ PCR/ RT-PCR protocol is necessary only in the indirect method, where the amplied product is present in the cells in the form of doublestranded DNA. Its purpose is the visualization of this amplied product by a specic detection method, namely, in situ hybridization.
In the direct method, the label is incorporated during the synthesis of the amplied product. This method can be used without amplication to visualize DNA or RNA in situ.
6.1 OVERVIEW
Two labeled probes, each of which is complementary to one of the two strands of neosynthesized DNA, hybridize to form labeled hybrids. The hybridization reaction does not require any input of energy. Hybridization is the basis of the RT and PCR steps (see Chapters 4 and 5). This is always the case for molecular hybridization. The opposite of hybridization is denaturation, which occurs at the melting point, and consists of using heat to separate the two strands of the hybrid. There are two bonds between A and T, and three between G and C.
It is due to the formation of hydrogen bonds between the different bases that make up the sequences of one of the strands of the amplied product and the probe that is complementary and anti-sense to it. The fact that the probe is labeled means that the complex or hybrid is also labeled, and this allows it to be detected.
For the labeling of the probe, see Section 6.3.3. For the detection process, see Chapter 7. 123
Hybridization
3'
5'
5'
3'
1
5' 3' 3' 5'
x Denaturation of the amplied DNA
2
5' 5' 3' 3' 3' 5' 5' 3'
y Hybridization of the two labeled probes on the two strands of DNA
z Labeled hybrids
Figure 6.1 The hybridization step.

Denaturation
Labeling the probes
Hybridization
Washes
Labeled hybrids
124
6.3
Tools: The Probes
6.3 TOOLS: THE PROBES

A probe is a nucleic acid whose sequence is determined by: Complementarity Anti-sense Specicity Two probes, anti-sense and sense, are necessary, given that the amplied product is double stranded. Of each strand of the amplied product
Hybridization can, in fact, be carried out with a single probe, but in this case the detection will be reduced by half.
6.3.1 Types of Probes

The nature of the probes is not a limiting factor as such. However, simplicity should be favored, which is the reason the most widely used probes are: Synthetic oligonucleotides cDNA sequences These are easily obtained and labeled, and give efcient hybridization. These consist of double strands of DNA, each of which can hybridize with one of the two strands of the amplied product. They are most often produced by a PCR in the liquid phase. A nested PCR using the amplied product can give smaller-sized probes. These are used only exceptionally.
cRNA
6.3.2 Characteristics
The characteristics of the probes must satisfy the following criteria: Complementarity Anti-sense Specicity 6.3.2.1 Complementarity For primers, specicity is not crucial [poly (A)], but for probes it must be carefully checked.
The sequence of the two strands of neosynthesized DNA is known, and the structure of the complementary sequences is identical to that of primers. 6.3.2.2 Anti-sense
See Section 4.3.1.
For hybridization to take place, the sequence of the probe must be anti-sense to the nucleotide sequence to which it is complementary.
See Section 4.3.1.
125
Hybridization 6.3.2.3 Specicity It is imperative that data banks be consulted to check that there is no homology between the sequences of the probes and the possible DNA or RNA sequences that may be present in the cell.
It is the specicity of the probes that denes the specicity of the indirect in situ PCR/RT-PCR method.
The specicity criterion only concerns the nature of the probes. 6.3.2.3.1 OLIGONUCLEOTIDE PROBES As is the case for primers, the synthesis of probes must satisfy certain criteria: Length It must be between 20 and 30 mers. Small nucleotide fragments may hybridize in a nonspecic way. HPLC (high-performance liquid chromatography) purication is recommended. Being complementary to one of the strands of the amplied product, a probe must not contain any palindromic sequences or interprobe complementarity, if nonspecic hybridizations are to be avoided. The two probes must contain very similar percentage of GC. In any case it should be <55%. See Section 4.3.1.3. This is similar for the different probes. A difference of >5C could favor the nonspecic hybridization of one of the probes. The position should be internal to the amplied product. It should not overlap the primers. The presence of primers in the cells after the washing step could result in a nonspecic signal. Each probe should be checked against a genomic data bank by analysis and comparison.
Sequence
Composition
Melting temperature (Tm)
Position
Checking
6.3.2.3.2 CDNA PROBES The cDNA sequence must be included in that of the amplied product. This cDNA can be obtained by: Amplication after insertion into a vector, or Synthesis by amplication of the amplied product This method takes a long time, and is seldom, if ever, used. It is easy to obtain the amplied product by PCR in the liquid phase, then to use a nested PCR to amplify a part of it, which can then be used as a cDNA probe.
126
6.3
Tools: The Probes
6.3.3 Labeling the Probes

To detect the hybrid, the probe carries a label that can be revealed. The labeling method depends on the nature of the probe: Oligonucleotide By 3 extension. This is the most commonly used method. By 5 extension (see Section 5.3.2.7). This method, using a radioactive nucleotide in the position, is rare, although it can be used to quantify amplication. With a single radioactive atom attached to the 5 end, the emitted radiation is proportional to the number of hybrids formed, and thus the number of copies obtained. By random priming or nick translation, if the cDNA is obtained after the insertion of a plasmid. By PCR with labeled primers and, more particularly, by nested PCR if the amplied product was also obtained by PCR in the liquid phase. By in vitro transcription. This method is not used after in situ PCR/RT-PCR.
cDNA
cRNA Only the two most generally used labeling methods will be presented here, namely: Labeling by PCR By 3 extension The labels are: Antigens, or Radioactive isotopes 6.3.3.1 Antigenic labels
Carried by nucleotides Biotin, digoxigenin, or uorescein 35 33 Generally S or P
These are carried by dUTP, and are the most widely used labels. Advantages Stability Easy to use Rapid detection process Resolution
See Section 5.3.1.3.1.
Easy to store the labeled nucleotides and probes No radioprotection measures necessary Numerous detection methods Immunohistochemical reaction Cellular
127
Hybridization Disadvantages There is a limit to the number of nucleotides that can be incorporated during labeling. Without precautions (e.g., the addition of unlabeled nucleotides), only one or two labeled nucleotides can be incorporated. This type of mixed extension can be extremely long, i.e., up to several times that of the probe itself. Except for probes whose uorescent labeling can be observed directly, this check requires a stained reaction (see Section 6.3.3.6). This is due to the steric size. Stained reactions, using a chromogen, are not quantiable.
The labeling of the probe needs to be checked. Hybridization is limited. The quantication of the signal is a delicate operation. 6.3.3.2
35 33
Radioactive labels See Figure 6.10. Only the phosphate in the position is incorporated into the polymer. This is the most commonly used label. This is long enough for the probe to be used before radiolysis occurs. Emission of particles at a level similar 33 to that of P, allowing good localization of the signal. 33 Similar to that given by P 33 Similar to that given by P Specic activity: 1500 Ci/mmol
The two main ones are: S P
The isotope is substituted in the position of the nucleotide. 6.3.3.2.1 S Its characteristics are: Half-life: 87.4 days Emission energy: 0.167 MeV Resolution: 10 to 15 m Sensitivity: Medium Autoradiographic efciency: 0.5 grain/ emission Advantages A less energetic isotope An excellent compromise Disadvantages The nucleotide is modied (substitution of an oxygen molecule in the phosphate group). There is a risk of oxidation.
35 35
The handling of S does not require onerous radioprotection measures. This is between sensitivity and efciency.
35
The chemical bond is unstable. It necessitates the use of protection (e.g., DTT, mercaptoethanol). It can bring about chemical modications in the molecule, thus causing background.
S is difcult to use.
128
6.3 6.3.3.2.2 P 33 32 The P label has all the advantages of the P label, and only minor disadvantages. Half-life: 25.4 days Emission energy: 0.25 MeV Resolution: 15 to 20 m Sensitivity: Medium Autoradiographic efciency Short exposure time 35 Close to that of S Good 35 Close to that of S 35 Close to that of S
33
Tools: The Probes
Advantages Low emission energy A short half-life No modication of the nucleotide Disadvantages Cost Short half-life 6.3.3.3 Labeling by PCR See Section 5.3.2.6. This kind of direct PCR gives a yield that is inversely proportional to the percentage of labeled nucleotides (close to 100%). The primers are situated on the sequence of the amplied product such that a smaller nested PCR fragment is generated. This method is recommended because of its specicity. This method is of no practical use, and in fact it presents the additional risk of a nonspecic attachment of the primers. PCR in the liquid phase, using the same primers as in situ PCR. x Obtaining the amplied product
5 3 2
Few radioprotection problems Short exposure times It is a physiological label Still high Difcult to store, although quick to use
6.3.3.3.1 OVERVIEW This is a direct PCR in the liquid phase, which incorporates labeled nucleotides during the amplication step. The amplication should give a labeled probe that hybridizes in situ on a sequence of the amplied product. The probe is obtained from: The amplied product derived from the PCR in the liquid phase, or Genomic DNA
5 5 3 1 3 5 3 3 5
3 5
y Hybridization of the primers, and extension by Taq DNA polymerase
129
Hybridization
3 5 5 3 5 3 5 5 3
Each of the two primers hybridizes to one of the strands of the amplied product. Nucleotides that carry the label, and unlabeled nucleotides.
3
5 3 3
z Incorporation of the labeled nucleotides into the neosynthesized strands Double-strand probe labeled at the end of the rst cycle.
{ Amplication The amount of probe obtained is proportional to the number of cycles. Figure 6.2 Obtaining a probe labeled by PCR. Advantages Specicity of the probes Simultaneous synthesis and labeling of the two probes Production of a large quantity of labeled probes Labeling along the entire length of the probes Size of the probes Disadvantages The usual difculties involved in using radioactive nucleotides That the two probes are entirely complementary to each other The size of the probes 6.3.3.3.2 PROTOCOL There is the risk of contamination of the PCR apparatus. They are PCR products. In this case, there is a high level of denaturation. This can be a disadvantage if the amplied fragment is very long. For reasons of simplication, only the labeling protocol using dNTPs conjugated to an antigen is presented here. Standard equipment They are made directly from the sequence of interest. This is the basic principle of PCR. The number of cycles is the only limiting factor. The density of the labeling depends on the ratio of the labeled nucleotide to the total amount of nucleotide. It can be up to 200 or 300 bp, i.e., practically the size of the amplied product to be detected.
Equipment Liquid PCR thermocycler 130
6.3 Reagents Sense and anti-sense primers dUTP-X-antigen Unlabeled dNTPs Taq DNA polymerase Mineral oil KCl MgCl2 TrisHCl Solutions
Tools: The Probes
Storage in aliquots at 20C Storage at 20C Storage at 20C Storage at 20C For PCR Molecular-biology grade Molecular-biology grade Molecular-biology grade
Sense and anti-sense primers 0.7 mM dUTP-X-antigen 2 mM unlabeled dNTPs Taq DNA polymerase 25 mM MgCl2 10X buffer
Sterile water 6.3.3.3.3

LABELING
All the solutions must be prepared using DNase-free reagents in a sterile container (see Appendix A1.1). 0.1 to 1 M (stored in aliquots at 20C) Addition of 1.3 mM dTTP Storage at 20C 5 U/l (storage at 20C) See Appendix B2.12. 100 mM TrisHCl; 500 mM KCl; pH 8.3 Storage in aliquots at 20C See Appendix B1.1.
ANTIGENIC
REACTION
MIXTURE
FOR
a. Place the following reagents in a sterile Eppendorf tube: Amplied product X Primers 250 nmol dATP, dGTP, dCTP X l Labeled dUTP X l Unlabeled dTTP X l MgCl2 210 l 10X buffer 5 l Taq DNA polymerase 1.5 U H2O b. Mix and centrifuge. c. Cover with oil. d. Place in the thermocycler. 6.3.3.3.4 PCR PROTOCOL 7 min 94C 1 min 60C 1 min 72C To 50 l 100 l
To be determined 2 mM 0.7 mM 1.3 mM To be determined
To be determined (0.5 to 2.5 U) Volume according to the concentration
First cycle Denaturation Hybridization Extension
The temperature varies according to the primer. The time can be extended if the probe to be synthesized is long. 131
Hybridization Following cycles (n cycles) The number depends on the required quann tity of probe: Q = q 2 , where Q is the nal quantity of probe, q is the initial quantity of probe, and n the number of cycles. 1 min 94C 1 min 60C 1 min 72C For large numbers of cycles, it is sometimes necessary to reduce the time to preserve the efciency of the enzyme. The temperature varies according to the primer. After 10 to 20 cycles, the time is generally increased to compensate for the loss in efciency of the enzyme.
Denaturation
Hybridization Extension Last cycle Denaturation Hybridization Extension Following step Precipitation with ethanol 6.3.3.4 Labeling by 3 extension
1 min 94C 1 min 60C 10 min 72C
In this cycle the time needs to be longer than in the previous cycles so that the extension of the newly formed strands can be completed. See Section 6.3.3.5.
This labeling method is used with oligonucleotides. 6.3.3.4.1 OVERVIEW The labeling of an oligonucleotide (<50 mers) is carried out by the addition of labeled nucleotides at the 3 end using an enzyme, i.e., terminal deoxynucleotidyl transferase (TdT). This requires a free 3 OH and nucleotide triphosphates. In the presence of cobalt ions, the enzyme (TdT) catalyzes the polymerization of a labeled deoxyribonucleotide. Random priming and PCR are not applicable to these oligonucleotides. In the case of antigenic nucleotides it is possible to add labeled and unlabeled nucleotides alternately, to obtain a longer extension. Cobalt is the cofactor of TdT.
132
6.3
1 5 2
TdT
Tools: The Probes
x Synthesized oligonucleotide probe (the 3 end must be hydroxylated).

3
y Enzymatic action: The terminal deoxynucleotidyl transferase (TdT) attaches itself to the 3 end, which contains a free OH.
z The addition of labeled dUTP and unlabeled dATPs, and polymerization: The TdT enzyme catalyzes the polymerization of the labeled deoxynucleotide triphosphates.
3
Figure 6.3 Labeling by 3 extension.
Advantages Radioactive or antigenic labels are used. The two probes cannot hybridize with each other. Quantication is possible. The regions to be hybridized can be located anywhere on the sequence of the product to be amplied. Reassociation with the probes is not possible. Storage is possible in the case of antigenic labeling. Each hybrid corresponds to a target nucleic acid. Given their small size, these probes may attach to damaged cell structures, thus increasing the risk of background.
This is a rapid, simple labeling method. It is possible to store the labeled probe. Quantication is possible. Disadvantage There is a possibility of the labeled probes diffusing.
6.3.3.4.2 EQUIPMENT/REAGENTS/SOLUTIONS Equipment Incubator at 37C Reagents Potassium cacodylate Cobalt chloride (CoCl2) Antigenic dUTP labels Biotin-X-dUTP Digoxigenin-X-dUTP, or Fluorescein-X-dUTP
Enzymatic reaction Molecular-biology grade To be used only for in situ hybridization Labeling kit
133
Hybridization Radioactive dATP labels Essentially ( S or P)-dATP; other radioisotopes are little used. The use of other dNTPs can lead to nonspecic signals. 25 to 45 nucleotides (mers); 30 nucleotides is a good average. Storage is at 20C. The enzyme is often sold with the appropriate buffer and CoCl2.
35 33
Oligonucleotides
Terminal deoxynucleotidyl transferase (TdT)
Solutions
CoCl2 25 mM Antigenic deoxynucleotides 1 mM biotin-X-dUTP 1 mM digoxigenin-X-dUTP 1 mM uorescein-X-dUTP Radioactive deoxynucleotides 35 33 ( S or P)-dATP
Water 2 to 100 pmol/l oligonucleotides Labeling (tailing) 5X buffer
50 U/l terminal deoxynucleotidyl transferase (TdT)
All the solutions are prepared using DNase- and RNase-free reagents in a sterile container (see Appendix A1.1). Storage at 20C (see Appendix B2.3.2) Storage at 20C See Figure 6.4. See Figure 6.5. See Figure 6.6. 35 33 The radioisotope ( S or P) occupies the position (see Figure 6.10) for labeling by 3 extension. Storage in aliquots at 80C or 4C Specic activity 3000 Ci/mmol Treated with DEPC (see Appendix A1.2) For labeling 1 M potassium cacodylate; 125 mM Tris HCl; 1.25 mg/l BSA; pH 6.6 Storage at 20C 200 mM potassium cacodylate; 1 mM EDTA; 200 mM KCl; 0.2 mg BSA; 50% glycerol (v/v); pH 6.5 Storage at 20C. The presence of glycerol means that the enzyme solution remains liquid at 20C. The enzyme is labile. Labeled oligonucleotides: 35 S-dATP 33 P-dATP Eppendorf type
6.3.3.4.3
PROTOCOL FOR RADIOACTIVE PROBES
Reaction mixture Place the following reagents in a sterile tube in the order indicated: Sterile water 10 pmol or 100 pmol oligonucleotides 5X reaction buffer 25 mM CoCl2 10 pmol labeled dATP 50 U/l TdT 5 l 5 l 4 l 4 l 1 l 1 l
For a nal volume of 20 l According to requirements If a red precipitate forms, need to check the origin of the radioactive nucleotide 35 50 Ci in the case of the S-dATP probe A labile enzyme; must not be allowed to rise to room temperature
134
6.3 Incubation 60 min 37C
Tools: The Probes
The labeling is carried out at 37C (30 min minimum). It is not necessary to prolong the incubation time, or to increase the amount of enzyme, because the 3 extension remains limited (see Section 6.3.3.6). An optional step
Stopping the reaction Incubate In a water bath, or By heating Following step Precipitation with ethanol 6.3.3.4.4
4C 10 min 65C
See Section 6.3.3.5. (biotin, digoxigenin, or uorescein)-dUTP
PROTOCOL FOR ANTIGENIC PROBES
Reaction mixture Place the following reagents in a sterile tube, in the order indicated: Sterile water 10 pmol or 100 pmol oligonucleotides 5X reaction buffer 25 mM CoCl2 1 mM labeled dUTP 10 mM dATP 50 U/l TdT Incubation Stopping the reaction In a water bath Following step Precipitation 6.3.3.5 Purication 4 l 5 l 4 l 4 l 1 l 1 l 1 l
Eppendorf type For a nal volume of 20 l Oligonucleotides diluted in sterile water, and stored at 20C Only one or two labeled nucleotides added at the 3 end The addition of unlabeled dATP, which allows the incorporation of several labeled dUTPs Catalyzes the polymerization of nucleotides at the 3 end A labile reagent, which must not be reheated Not necessary to prolong the incubation time, or to increase the amount of enzyme, given that the extension is limited An optional step, which can be replaced by the addition of EDTA (2 l), or by heat (65C for 10 min) The oligonucleotides are puried by ethanol precipitation (see Section 6.3.3.5).
60 min 37C 4C
6.3.3.5.1 OVERVIEW Nucleic acids are water soluble, and precipitate in an alcoholic solution in the presence of salts.
Alternatively, nucleic acids can be separated from free nucleotides and labeling reagents by passage through a column.
135
Hybridization 6.3.3.5.2 EQUIPMENT/REAGENTS/SOLUTIONS Equipment Centrifuge Vacuum jar or Speedvac Freezer (20C or 80C) Solutions
14,000 g To dry the probe To store reagents and precipitation products All the solutions should be prepared using DNase- and RNase-free reagents in a sterile container (see Appendix A1.1). See Appendix B2.18. See Appendix B.2.2. See Appendix B2.15. Storage at 20C Storage at 20C
3 M sodium acetate; pH 5.2 7.5 M ammonium acetate; pH 5.5 10 mg/ml transfer RNA (tRNA) 4 M lithium chloride Ethanol, 100 and 70
6.3.3.5.3 PROTOCOL Reaction mixture a. On ice, add the reagents in the following order: 10 mg/ml tRNA 2 l 7.5 M ammonium acetate, or 3 M sodium acetate, or 4 M lithium chloride 1/5 the nal volume 23 vol
Ethanol 100 b. Vortex, centrifuge. Incubation a. Incubate.
This facilitates the precipitation of oligonucleotide probes; optional. The ammonium acetate can be replaced by 3 M sodium acetate or 4 M lithium chloride. In the latter case, the pellet must be washed with 70 alcohol (stored at 20C) after precipitation and recentrifugation. Store at 20C. The volume of the reaction solution No reagent must remain on the side of the tube. Precipitation of the probe Minimum 30 min at 80C, or Minimum 2 h at 20C With the tube oriented to facilitate the localization of the pellet and the removal of the supernatant May contain radioactive nucleotides
b. Centrifuge. Washing a. Remove the supernatant. b. Wash the precipitate. Ethanol 70 c. Centrifuge.
60 min 80C or overnight 20C >14,000 g 15 min 4C
50 l >14,000 g 15 min 4C
To eliminate salts With the tube oriented to facilitate the localization of the pellet and the removal of the supernatant
d. Remove the supernatant. e. Dry. 136
6.3 6.3.3.6 Checking/storage/utilization
Tools: The Probes
Before a probe is used or placed in storage, its labeling must be checked. 6.3.3.6.1 CHECKING THE LABELING The type of check depends on the label: Antigenic labels Fluorescent labels Radioactive labels The length of a labeled probe can be checked by the use of an agarose gel. An immunohistochemical reaction is used to reveal a range of dilutions of the labeled probe on a nylon membrane. The detection of the uorescence after deposition of a range of dilutions on a nylon membrane exposed to ultraviolet radiation. This is a measure of the specic activity of the probe. This check needs to be carried out only if the background level is high. The specicity of small fragments is very limited.
6.3.3.6.2 STORAGE This depends on the nature of the label: Antigenic probes
Radioactive probes
Antigenic labels are very stable. Solubilize in TrisEDTA buffer (see Appendix B3.6). Store at 20C for several months. At 80C, the risk of radiolysis is limited. The half-life of the radioelements: 33 P: half-life 25.4 days; storage 1 week 35 S: half-life 87.4 days; storage <1 month Store in a dry state, or in sterile water at 80C. Oligonucleotide probes can be used directly. If the probe is labeled with S, add 10 mM DTT.
35
6.3.3.6.3 UTILIZATION Double-strand probes (i.e., obtained by PCR) have to be denatured before hybridization. a. Denature. 10 min 92C or 5 min 96C b. Cool quickly in ice. 0C c. Utilize.
At this temperature, the two strands cannot rehybridize. The probe can be denatured in the hybridization reaction medium.
137
Hybridization
6.4 HYBRIDIZATION PARAMETERS

The specicity of the hybridization reaction depends on the factors that limit the nonspecic binding of the probes. Specicity is very important in the indirect PCR/RT-PCR method, in that it limits the kind of stringent washing which is needed to eliminate nonspecic hybrids, but which can also damage tissue or cell structures that are already in a delicate state. The higher the temperature, the more specic the hybridization will be. Above Tm (see Section 4.3.1.3), 50% of the specic hybrids are denatured. The salt concentration is proportional to the stability of specic and nonspecic hybrids. Its pH affects the stability of the hybrid. DNADNA hybrids are less stable than DNARNA hybrids. The longer the probe, the more specic the hybrid.
Hybridization temperature
Salinity Hybridization buffer Type of probe Probe length
6.4.1 Hybridization Temperature

This is the temperature at which the hybridization reaction is carried out. The sections incubate in the presence of the reaction medium, in a moisture chamber, at the temperature where the lowest possible number of nonspecic hybrids is formed. This temperature depends on the Tm of the probes. 6.4.1.1 Melting temperature (Tm) See Section 4.3.1.3. This acts as a check that the Tm of the chosen pair of probes are mutually compatible.
The formation of hybrids is a spontaneous reaction. Tm = the temperature at which 50% of specic hybrids are denatured (see Section 4.3.1.3).
The Tm is calculated using formulae that are precisely adapted to the nature of the probe. The calculation of the Tm can be carried out automatically by certain types of software on the basis of the probe sequences. 6.4.1.2 The difference between Tm and the hybridization temperature
The hybridization temperature is determined empirically, and depends on the nature of the probe: cDNA probe The hybridization temperature is 10C below the Tm. Oligonucleotide probe The hybridization temperature is 5C below the Tm. 138
If the hybridization temperature is too far below the Tm, a large number of hybrids will form, but their specicity will be reduced. 200 to 300 bp 20 to 30 mers
6.4 The hybridization temperature can be articially lowered by the addition of formamide. The inuence of temperature on the hybridization process can be illustrated as follows:
100 C Tm 37C 0C -
Hybridization Parameters
The addition of 1% formamide lowers the hybridization temperature by 0.65C.
At room temperature, and up to 37C, the DNA matrix is double stranded.

1
100 C Tm 37C 0C -
Between 94 and 100C, denaturation takes place, i.e., the two strands separate out.
100 C Tm 37C 0C -
If, after denaturation, the single-stranded DNA is immediately brought down to a temperature close to 0C, no hybrid will form. If the hybridization temperature is close to room temperature, a large number of hybrids, both specic and nonspecic, will form.
100 C Tm 37C 0C -
100 C Tm 37C 0C -
The ideal hybridization temperature, i.e., the one at which the maximum number of specic hybrids will be formed, is 5 to 10C below the Tm.
100 C Tm 37C 0C -
If the hybridization temperature is the same as the Tm, only 50% of specic hybrids will form (this follows from the definition of Tm).
100 C Tm 37C 0C -
Finally, if the hybridization temperature reaches 94 to 100C, no hybrids will form, as the nucleic acids are denatured.
7
Figure 6.4 Determination of the hybridization temperature. 139
Hybridization
6.4.2 Na Ion Concentration

The Na ion concentration inuences the stability of the hybrids. If it is raised tenfold, the Tm will rise by 16.6C, which means that the hybrids will be more stable.
+
Salinity The most common concentration is around 660 mM. This corresponds to 4X SSC buffer (see Appendix B3.5).
6.4.3 Hybridization Buffer

This limits variations in the pH. Large variations in the pH destabilize interbase hydrogen bonds.
6.4.4 The Nature of the Probes

Since DNARNA hybrids are more stable than DNADNA hybrids, it may be necessary, in very rare cases, to use RNA probes. This type of hybridization does not take place, in practice, after in situ PCR/RT-PCR.
6.4.5 Probe Length

The longer the probe, the higher the specicity and stability of the hybrids. Labeling by the 3 extension of an oligonucleotide probe can give a probe that is two to three times longer than the original probe. The Tm rises with the length of the probe. This fact must be taken into account when calculating the hybridization temperature.
6.4.6 Duration
Most authors agree that the reaction is optimal after 3 h of incubation. 316 h The reaction can be continued for up to 16 h (i.e., overnight) without any risk for the hybrids. Morphology, however, deteriorates over time.
6.5 PROTOCOL
6.5.1 Equipment/Solutions
6.5.1.1 Equipment Sterilized, wrapped in aluminum foil, and stored at room temperature; gloves must be worn when handling them Of the petri box type (24 24 cm), with enough space for the slides
Cover slides 12 12 mm cover slides 24 50 mm cover slides 9 mm circular cover slides Moisture chamber
140
6.5 Oven Thermocycler Vortex mixer 6.5.1.2 Solutions
Protocol
37 to 65C Can also be used for the hybridization step
Alcohol 70, 95, and 100 Deionized formamide
50X Denhardts solution
50% dextran sulfate 1 M dithiothreitiol
10 mg/ml DNA
0.5 mg/ml heparin 10 mg/ml poly (A) 10 mg/ml RNA
Sarcosyl 20X SSC (standard saline citrate), pH 7.0 Sterilized water
TE buffer/NaCl
See Appendix B2.4. The formamide must be of good quality (very pure) and deionized (to maintain the pH of the buffer). Storage is at 20C in aliquots of 500 l. If the reagent is not frozen at 20C, it should not be used. See Appendix B2.5. Storage is at 20C in aliquots. Unfreezing several times is possible without modication of its properties. See Appendix B2.6. Storage is at 20C. See Appendix B2.7. Storage is at 20C in aliquots of 50 to 100 l. It cannot be refrozen. Its nauseous odor is the best guarantee of its condition. It should not be used if the odor is modied or absent. See Appendix B2.8. Sonication Storage is at 20C. The freezing/unfreezing cycles can cause breaks. Storage is at 20C. See Appendix B2.13; equivalent to RNA. See Appendix B2.15. Storage is at 20C. The freezing/unfreezing cycles can cause breaks. See Appendix B2.17. See Appendix B3.5. Storage is at 4C, or at room temperature. See Appendix B1.1. Use only immediately upon opening the bottle, or use DEPC-treated water (see Appendix B1.2). See Appendix B3.6.2.
6.5.2 The Reaction Medium

The reaction medium is made up of the hybridization buffer plus the two probes. The hybridization buffer can be stored, at least for a few weeks, at 20C, either alone or added to the probe, without losing any of its effectiveness.
141
Hybridization Hybridization buffer 20X SSC buffer
Deionized formamide
50%
50% dextran sulfate, or
10%
0.5 mg/ml heparin 10 mg/ml RNA, or 10 mg/ml poly (A) 10 mg/ml DNA 50X Denhardts solution 1 M DTT
0.1 mg/ml 100250 g/ml 100 g/ml 100400 g/ml 12X 10 mM
Hybrids form at pH close to neutrality in the + presence of Na ions (ionic strength). + The Na ion concentration is of major signicance for the stability of the hybrid, on which it has a direct effect. This is Indispensable. The addition of formamide reduces the hybridization temperature (see Appendix B2.4). This increases the effectiveness of the hybridization process by lowering the Tm. This increases the effectiveness of the hybridization reaction, and also speeds it. It is a nonionic polymer that concentrates the solute through the volume that it occupies in the solution. This protects cellular RNA. This can replace dextran sulfate. This saturates nonspecic bonds of the proteic type by competition. This saturates nonspecic bonds; can be replaced by a poly (C) or a poly (G). This saturates nonspecic bonds. This saturates nonspecic bonds by competition with macromolecules. This is used only with probes labeled with 35 S (it is an antioxidant against the radiolysis 35 of S). This is used only after denaturation (3 min at 100C), since it is destroyed at 100C. Add up to the saturating concentration, i.e., the concentration at which no further addition of probe will modify the intensity of the signal. After centrifugation and drying, the labeled probe is dissolved directly in the hybridization buffer.
Reaction medium a. Add the probe to the hybridization buffer. 0.10.2 g/ml hybridization buffer 110 g/ml hybridization buffer
Radioactive probe Antigenic probe b. Vortex. c. Centrifuge.
6.5.3 The Different Steps

a. Place the reaction medium on the tissue sections. 142 3050 l/ section
6.6 b. Cover the sections with: cover slides, or Cover clips 3 min 100C d. Place the slides directly in the moisture chamber. c. Denature.
Posthybridization Treatments
If the sections are hybridized in a moisture chamber If the sections are hybridized in a thermocycler Indispensable, since the amplication product is double-stranded DNA With 5X SSC buffer, in the incubator programmed for the chosen temperature. Possible also to program the thermocycler for this step More generally, overnight
e. Incubate.
At least 3 h, and up to 16 h
6.6 POSTHYBRIDIZATION TREATMENTS

6.6.1 Aim
The aim is to eliminate as many nonspecic hybrids as possible, while conserving the maximum number of specic hybrids. During the hybridization step, the probes x with a very high degree of afnity to perfectly homologous nucleic acid molecules, but also, with a lesser degree of afnity, to nucleic sequences whose homology is only partial. The purpose of posthybridization treatments (e.g., washes) is to eliminate nonspecic hybridization, which introduces nonspecic signals and masks specic hybridization by reducing the signal to noise ratio.
Matched hybrid Mismatched hybrid A succession of washes is carried out in increasingly stringent conditions so that only specic hybrids, which are the most stable, remain. Furthermore, a probe tends to x in a totally nonspecic way to non-nucleic structures, essentially those of proteins.
6.6.2 Parameters
The parameters that determine the degree of stringency, and thus the choice of hybrids (in terms of their stability), are: Temperature Ionic strength Washing time The more stringent the conditions, the more the hybrids are denatured. Mismatched hybrids are denatured before matched hybrids.
143
Hybridization 6.6.2.1 Temperature This is the most important factor for the stability of hybrids. This means the disappearance of specic hybrids. For in situ hybridization, the washing temperature (Tw) is determined by washing with 2X SSC at temperatures varying by 5C between 40 and 70C.
The higher the temperature, the higher the risk of denaturation. Only internal control makes it possible to determine the optimal washing temperature. The presence of negative and positive cells conrms the disappearance of nonspecic hybrids. 6.6.2.2 Na ion concentration
+ +
The salt [Na ] concentration is proportional to the stability of the hybrids. Washing in decreas+ ing concentrations of Na ions brings about the dissociation of the hybrids; the less stable are denatured before the more stable. 6.6.2.3 Washing time
Mismatched hybrids and nucleic acid protein bonds are less stable than specic hybrids. Washing in water would bring about complete denaturation.
Little difference is observable after an hour.
Ceteris paribus, repeated washes are more effective than long washes.
Washing with moderate shaking gives the best reproducibility of results. Washing accompanied by intense or prolonged shaking can cause damage to the sections, and is more difcult to reproduce. Limiting the number of washes can be compensated for by voluminous washes accompanied by shaking.
6.6.3 Protocol
5X SSC 5 min For the rst rinse, which serves only to unstick the cover slides, place the slides in a borrel tube or tray containing the solution, and let the cover slide off gently without touching it. Necessary Washing temperatures, to be determined Room Temperature An optional step. Temperature is a factor + of stringency that is very effective at this Na concentration. Use a water bath with shaking. An optional step. To be used only with + radioactive probes. The Na concentration is very low, and can cause dissociation of specic hybrids.
2X SSC 2X SSC 1X SSC 0.5X SSC
30 min Tw 30 min rt 30 min rt 30 min rt 30 min rt
0.1X SSC
144
6.7
Before Revelation
6.7 BEFORE REVELATION

Depending on whether the label is Radioactive or Antigenic the preparation will be different. At this stage, the probe label is the hybrid label.
6.7.1 Radioactive Hybrids

6.7.1.1 Dehydration 1 rapid
The sections must be dried before contact with autoradiographic lm or emulsion.
Alcohol 70
It is advisable to make up the solutions of alcohol with 300 mM ammonium acetate to maintain the ionic strength. This salt is volatile, so it does not cause deposits during the drying of the sections.
Alcohol 95 Alcohol 100 6.7.1.2 Drying
1 rapid 1 rapid Indispensable 3060 min The aim of this step is to eliminate all trace of alcohol and water (especially from tissue), which could cause background noise on macroautoradiograms. See Section 7.3.3. See Section 7.3.4.
In a vacuum jar
Following steps Contact with autoradiographic lm Dipping in the autoradiographic emulsion
6.7.2 Antigenic labels

For immunohistological detection, do not dry tissue sections. The tissue is transferred to the buffer that will be used during the detection process. Following step Immunohistochemical detection Indispensable
See Section 7.2.
145
Chapter 7
Revelation
Contents
CONTENTS
7.1 7.2 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunohistochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.1 Immunohistochemical Reaction . . . . . . . . . . . . . . . . . . . . . . 7.2.1.1 Immunocytochemical Tools . . . . . . . . . . . . . . . . . 7.2.1.1.1 Antibodies . . . . . . . . . . . . . . . . . . . . . . 7.2.1.1.2 Streptavidin . . . . . . . . . . . . . . . . . . . . . 7.2.1.1.3 Biotin . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.1.2 Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.1.2.1 Enzymes . . . . . . . . . . . . . . . . . . . . . . . 7.2.1.2.2 Fluorescent Labels . . . . . . . . . . . . . . . 7.2.1.2.3 Particle Labels. . . . . . . . . . . . . . . . . . . 7.2.1.2.4 Conjugates. . . . . . . . . . . . . . . . . . . . . . Detection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.2.1 Direct Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.2.1.1 Overview. . . . . . . . . . . . . . . . . . . . . . . 7.2.2.1.2 Advantages/Disadvantages . . . . . . . . . 7.2.2.2 Indirect Reaction. . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.2.2.1 Overview. . . . . . . . . . . . . . . . . . . . . . . 7.2.2.2.2 Advantages/Disadvantages . . . . . . . . . 7.2.2.3 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.2.3.1 Solutions . . . . . . . . . . . . . . . . . . . . . . . 7.2.2.3.2 Direct Reaction Protocol. . . . . . . . . . . 7.2.2.3.3 Indirect Reaction Protocol . . . . . . . . . 7.2.2.3.4 Particular Case: The Biotin Label . . . . . . . . . . . . . . . . . Revelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.3.1 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.3.2 Alkaline Phosphatase . . . . . . . . . . . . . . . . . . . . . . 7.2.3.3 Peroxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 152 152 152 152 153 153 154 154 155 155 155 156 156 156 156 156 156 157 157 157 158 158 159 161 161 161 163 165 165 166 166 166 166 166 167 167 167 167 168 168 169 149
7.2.2
7.2.3
7.3 Autoradiography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.1 7.3.2 Principles of Autoradiography . . . . . . . . . . . . . . . . . . . . . . . Characteristics of Emulsions . . . . . . . . . . . . . . . . . . . . . . . . 7.3.2.1 Radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.2.2 Photographic Emulsions . . . . . . . . . . . . . . . . . . . . 7.3.2.3 Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.2.4 Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.2.5 Efciency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.2.6 Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.2.7 Artifacts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.2.8 Quantication . . . . . . . . . . . . . . . . . . . . . . . . . . . . Macro-Autoradiography . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.3.1 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.3.2 Autoradiographic Film . . . . . . . . . . . . . . . . . . . . .
7.3.3
Revelation Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.3.3.1 Equipment/Solutions. . . . . . . . . . . . . . 7.3.3.3.2 The Different Steps . . . . . . . . . . . . . . . Micro-Autoradiography . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.4.1 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.4.2 Types of Emulsion . . . . . . . . . . . . . . . . . . . . . . . . 7.3.4.3 Advantages/Disadvantages . . . . . . . . . . . . . . . . . . 7.3.4.4 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.4.4.1 Equipment/Reagents/Solutions . . . . . . 7.3.4.4.2 The Different Steps . . . . . . . . . . . . . . . 7.3.3.3 169 169 170 171 171 171 171 172 172 173
7.3.4
150
7.1 The choice of detection method depends on the hybrid label: Radioactive isotope Antigen There are two possible approaches, depending on the label: Immunohistochemistry
Diagram of the Different Steps
Detection of radiation Immunological detection
Autoradiography
Given the amplication necessary for a strong signal, whose interpretation and validity are sometimes questionable, preference is given to detection systems that increase the signal/background ratio and give good cellular resolution.
Immunohistochemistry involves an antigen antibody reaction, which is visualized by: A stained reaction, or Fluorescence Autoradiography involves the visualization of emitted radiation by the use of a photographic emulsion, either: A solid emulsion on lm, or A liquid emulsion, with which the tissue is coated What is sought is the cellular or subcellular location of DNA or RNA. Radioactive labeling does not provide enough accuracy, which is the reason antigenic labeling is much more highly favored.

To detect an antigenic hybrid, which comes from: The incorporation of a nucleotide coupled to an antigenic molecule, or A PCR or RT-PCR in the presence of a primer labeled with an antigenic molecule, or The hybridization of the amplied product by probes into which a haptene has been incorporated. During a direct PCR or RT-PCR See Section 5.3.1.3 See Section 5.3.2.7 See Section 6.3.3
Antigenic hybrids
Direct detection
Indirect detection
151
Revelation To detect a radioactive hybrid, which comes from: The incorporation of a radioactive nucleotide in a direct PCR, or Amplication in the presence of a radiolabeled primer, or The hybridization of the amplied product by radioactive probes [ S]dATP or dCTP This method is not recommended (see Section 5.3.1.3) 35 [ S]dATP or dCTP (see Section 5.3.2.7)
35
Radioactive hybrids
Macro-auto radiography
Micro-auto radiography
7.2 IMMUNOHISTOCHEMISTRY
The antigenic hybrid has one or more sites (i.e., hapten) where an antigenantibody complex can form during the immunohistological reaction. The most commonly used haptenes are: Biotin Digoxigenin Fluorescein (see Section 5.3.1.3.1)
7.2.1 Immunohistochemical Reaction

The purpose of this reaction is to form a complex of high afnity between the antigen and a molecule that is specic to it (a tool) to visualize the antigen, and thus the amplied products. 7.2.1.1 Immunocytochemical tools
These are molecules which have a high degree of afnity with haptenes. Antibody Streptavidin Such molecules are produced as a response to immunization by the haptene. This spontaneously forms a complex with biotin.
7.2.1.1.1
ANTIBODIES It is the G immunoglobulins (IgG) that are most widely used in immunohistology.
Immunoglobulin of polyclonal or monoclonal origin Fragments of IgG: Fab and F(ab)2 152
7.2
Immunohistochemistry
The F(ab)2 immunoglobulin fragment is obtained by enzymatic digestion (cleavage by pepsin) (variable sequence of Mw = 100 kDa), which, after reduction, can give rise to two Fab fragments with properties analogous to those of the Fab fragment. The Fab immunoglobulin fragment is obtained by enzymatic digestion (action of papain) (variable sequence). The Fc immunoglobulin fragment is obtained by enzymatic digestion (action of pepsin) (constant sequence). Figure 7.1 Molecule of IgG, and fragments resulting from its proteolytic digestion. 7.2.1.1.2 STREPTAVIDIN 15 Streptavidin has a very high afnity (Kd = 10 1 ) for biotin. One molecule of streptavidin can bind four biotin molecules by noncovalent binding.
This is a homotetramer isolated from Streptomyces avidinii. In comparison to avidin, its advantages are that: It is a nonglycosylated protein. It has a neutral isoelectric point. It does not interact with lectins. Similar molecules are available commercially, e.g., avidin and streptavidin: Avidin: a glycoprotein of low molecular mass obtained from egg white, which has four xation sites for biotin Extravidin
Figure 7.2 Streptavidinbiotin complex.
7.2.1.1.3 BIOTIN One of the properties of this vitamin is its very high afnity bond with streptavidin (see Figure 6.4).
Biotin is used as: A haptene (i.e., labeled nucleotides; see Section 5.3.1.3) A conjugated label (see Section 7.2.1.1) 153
Revelation It can be coupled with: IgG Fab, F(ab)2 fragments Streptavidin 7.2.1.2 Labels These labels are adsorbed onto either immunoglobulin or streptavidin. In this case it is not conjugated to a label, and must be considered as an antigen. Unsaturated complex.
Different types of label can be used: Enzymatic Fluorescent Particular 7.2.1.2.1 ENZYMES Some enzymes are more stable, and for this reason are more commonly used. Peroxidase Horseradish peroxidase (black radish extract) is an enzyme of Mw = 40 kDa. Advantages Numerous chromogens Multiple labeling Insoluble in alcohol Disadvantages Endogenous enzymes Diffusion of the precipitate Alkaline phosphatase Alkaline phosphatase (extracted from calf intestine) is a very widely used enzyme of Mw = 80 kDa. Advantages Numerous chromogenic substrates Sensitivity
Numerous chromogens exist for all the different enzymes. This is a small molecule.
Precipitates of different colors (see Section 7.2.3) Complementary to alkaline phosphatase The possibility of embedding in resin Pretreatment of sections by the addition of an inhibitory agent such as 10% hydrogen peroxide, combined or not with sodium nitrite or methanol (see Appendix B6.2.2) This is a large molecule. This enzyme is frequently found in biological tissue. Its endogenous activity is inhibited by levamisole or heat (see Appendix B6.2.1.2). Precipitates of different colors are available. The NBT-BCIP (see Appendix 6.1) system produces two precipitates with a single alkaline phosphatase molecule. Ready-to-use chromogenic systems are available. The conjugates are available in all forms (IgG, Fab, streptavidin, etc.). There is complementarity to peroxidase. Bright-eld microscopy is used.
Simplicity, reproducibility
Multiple labeling Ease of observation
154
7.2 Disadvantages Soluble in alcohol
Cannot be conserved Endogenous phosphatase Resolution less than that obtained with uorescent labels 7.2.1.2.2 FLUORESCENT LABELS A uorochrome is a molecule whose structure includes conjugated double bonds that, in response to excitation by a photon, emit another photon of a longer wavelength than that of the exciting photon. These labels are little used for detection purposes because of their low level of resolution and the lack of sensitivity of the methods associated with them.
This avoids dehydration. Embedding after dehydration is possible if the reaction is very intense (loss of signal, but also attenuation of the background). Chromogens are unstable over time. Conservation is possible at 4C. Pretreatment is indispensable to the inhibition of the signal (see Appendix B6.1.2). Colored precipitates are diffused around enzymatic sites. This structural property is responsible for the uorescence resulting from excitation by light. Observation necessitates a uorescence or confocal microscope. The signal cannot be conserved, in spite of any precautions that may be taken (antifading techniques, darkness, storage of slides at 4C). 1 to 20 nm See Section 8.13
7.2.1.2.3 PARTICLE LABELS Particles of colloidal gold can also be used, but their size means that latensication with silver is necessary if they are to be observed by light microscopy. 7.2.1.2.4 CONJUGATES
Because labeling is difcult to reproduce, it is better to use commercially available products. The experimental conditions for the preparation of labels, and their adsorption, necessitate specic conditions for each label and each tool. According to its size, a label can form a complex either with one or several immunoglobulins, or with their fragments.
Conjugates-antibodies Immunoglobulin, or Fragments can be conjugated with all the aforementioned labels.
Streptavidin conjugates The same conjugates are obtained with streptavidin. Biotin conjugates The low molecular mass of biotin means that several molecules can bind to each molecule of the label.
All the biotin conjugates are commercially available.
155
Revelation Biotinstreptavidin conjugates The concentration of conjugated biotin must not be saturating for the streptavidinbiotin binding sites.
See Figure 7.5.
7.2.2 Detection
The detection of an antigen contained in the amplied products (direct PCR/RT-PCR reaction) or the hybrids (indirect PCR/RT-PCR reaction) is obtained by an immunohistochemical reaction, either: Direct, or Indirect 7.2.2.1 Direct reaction See Chapter 1.
7.2.2.1.1 OVERVIEW The antigen is detected by a primary antibody (IgG), or fragments conjugated to a label. The haptene incorporated into the hybrid is detected by: x Fab fragments conjugated to a label y Immunoglobulins (primary antibodies) conjugated to a label
Figure 7.3 The direct immunohistochemical reaction. 7.2.2.1.2 ADVANTAGES/DISADVANTAGES A single step Which is nonetheless limited Essentially due to steric hindrance, and to the low level of antigenic labeling of the probes
Advantages Rapidity The possibility of amplication Disadvantage Low sensitivity
7.2.2.2
Indirect reaction The possibility of amplication
7.2.2.2.1 OVERVIEW The antigen is detected by a two-step reaction involving:
156
7.2 The formation of a complex with haptene by a succession of antigenantibody reactions, and A conjugated molecule antibody (IgG), or fragments conjugated to a label.
x The haptene incorporated into the hybrid is detected y By an anti-haptene derived from species X [IgG, Fab, F(ab)2], then z By an anti-species-X [IgG, Fab, F(ab)2], conjugated to a label Figure 7.4 The indirect immunohistochemical reaction. 7.2.2.2.2 ADVANTAGES/DISADVANTAGES Several secondary antibodies can x to the primary antibody. There is a cascade of antigenantibody reactions. There are successive incubations with each component.
Advantages Sensitivity > the direct method Amplication of the signal Disadvantage Long reaction time
7.2.2.3 7.2.2.3.1
Protocol SOLUTIONS Indirect reaction Monoclonal or polyclonal IgG Possible to use any label Direct reaction Indirect reaction
Antibodies Nonconjugated anti-haptene IgG IgG, F(ab)2, Fab Conjugated anti-haptene Anti-species X conjugate Inhibition of endogenous enzymatic activity Phosphatases Peroxidases Buffers Blocking buffers 50 mM TrisHCl buffer; 300 mM NaCl; 1% albumin serum 50 mM TrisHCl buffer; 300 mM NaCl; 2% goat serum 50 mM TrisHCl buffer, pH 7.6
See Appendix B6.2.1.2. See Appendix B6.2.1.3. See Appendix B6.2.1.1. Other agents can be added to the blocking buffer (see Appendix B6.2.1.1). For TrisHCl, see Appendix B3.7.1. 157
Revelation 50 mM TrisHCl buffer; 300 mM NaCl, pH 7.6 7.2.2.3.2 DIRECT REACTION PROTOCOL For TrisHCl/NaCl, see Appendix B3.7.5.
All the different steps are carried out at room temperature. 10 min This balances the osmolarity of the tissue after the posthybridization washing steps. Indispensable for eliminating any reaction at nonspecic sites, as the sections are preincubated with a nonspecic serum Either by aspiration or by carefully wiping the part of the slide around the tissue with lter paper Optional (see Appendix B6.2.1); the presence of endogenous enzymatic activity must be checked Formation of the hapteneantibody complex Primary antibody: (IgG), conjugated with Fab fragments (see Section 7.2.1.2) Dilution of the antibody always weak (1:10 to 1:100, according to the manufacturers instructions) Moisture chamber (water on lter paper) Can be increased if background See Section 7.2.3. See Chapter 11. All the following steps are carried out at room temperature. It is possible to make a circle of hydrophobic material to limit the quantity of solution spread on the section. 10 min This balances the osmolarity of the tissue after the posthybridization washing steps. This is an optional step for limiting any reaction at nonspecic sites. The sections are preincubated with a nonspecic serum. Optional (see Appendix B6.2.1). The presence of endogenous enzymatic activity must be checked.
a. Rinse. TrisHCl/NaCl buffer b. Block the nonspecic sites. Blocking buffer
1530 min
c. Eliminate excess buffer.
d. Inhibit endogenous enzyme.
e. Spread the conjugated antibody. Diluted in TrisHCl/NaCl buffer
20 l/ section
f. Incubate the antibody. g. Rinse. TrisHCl/NaCl buffer Following steps Detection Observation 7.2.2.3.3
2 h overnight 3 10 min
INDIRECT REACTION PROTOCOL
a. Rinse TrisHCl/NaCl buffer b. Block nonspecic sites. Blocking buffer
1530 min
c. Inhibit endogenous enzymatic activity.
158
7.2 d. Rinse TrisHCl/NaCl buffer e. Eliminate excess buffer.
3 10 min
f. Spread the antibody. Diluted in TrisHCl/NaCl 20 l/ buffer section g. Incubate the antibody. 6090 min h. Rinse TrisHCl/NaCl buffer 3 10 min i. Eliminate excess buffer. j. Spread the conjugated antibody. Diluted in TrisHCl/NaCl 20 l/ buffer section
There is a risk of dilution during the following step. This forms the hapteneantibody complex. Dilute to 1:50 to 1:500 (according to the manufacturers instructions). Moisture chamber (water on lter paper) Or TrisHCl buffer Detection of the complex Secondary antibody: IgG, Fab-fragments, or biotin conjugates, diluted to 1:25 to 1:50 (according to the manufacturers instructions) Dilution less than that of the rst antibody Moisture chamber (water on lter paper) On sections (100 l) or in trays
k. Incubate the antibody l. Rinse. TrisHCl/NaCl buffer m. Eliminate excess buffer. Following steps Detection Observation 7.2.2.3.4
6090 min 3 10 min
See Section 7.2.3. See Chapter 11.
PARTICULAR CASE: THE BIOTIN LABEL x Direct reaction Streptavidin is conjugated to a label.
y Indirect reaction The rst step uses native streptavidin, and in the second, the sites that are free of streptavidin are saturated with conjugated biotin. Conjugated streptavidinbiotin complexes are commercially available.
z Direct or indirect immunohistological reaction The reaction uses an anti-biotin IgG. This property is used to amplify the signal in the indirect reaction. Figure 7.5 The detection of biotin. 159
7.2 Advantages Sensitivity Specicity Numerous labels available Disadvantage Endogenous biotin
Due to the high afnity of streptavidin for biotin Multiple labelings This is present in some types of animal tissue (kidney, heart, muscle, liver), and must be inhibited.
Protocol Solutions Antibodies Anti-biotin IgG
Antispecies conjugated IgG Goat serum Streptavidin Conjugated streptavidinbiotin complex
Conjugated (direct immunohistochemical reaction) or nonconjugated (indirect immunohistochemical reaction) See Figure 7.4, indirect immunohistochemical reaction A nonspecic antibody that can be replaced by a blocking solution Conjugated or nonconjugated (see Figure 7.5) Possible to produce this complex in two steps on the section (see Figure 7.5), using: Nonconjugated streptavidin Conjugated biotin See Appendix B6.2.1.2. See Appendix B6.2.1.3. See Appendix B3. See Appendix B62.1.1. The purpose of the high concentration of + Na ions is to preserve the hybrids. It is possible to add other agents to the blocking solution. For TrisHCl buffer, see Appendix B3.7.1. For TrisHCl/NaCl buffer, see Appendix B3.7.5.
Inhibition of endogenous enzymatic activities: phosphatases peroxidases Buffers blocking buffers +50 mM TrisHCl buffer; 300 mM NaCl; 1% albumin serum +50 mM TrisHCl buffer; 2% goat serum; 0.1% Triton X-100 50 mM TrisHCl buffer, pH 7.6 50 mM TrisHCl buffer; 300 mM NaCl; pH 7.6 Streptavidin protocol a. Block nonspecic sites. Blocking buffer 10 min b. Form the biotinstreptavidin complex. Conjugated streptavidin 100 l/ diluted to 1:20 to 1:50 in section TrisHCl/NaCl buffer 6090 min
The dilution depends essentially on the label used. Streptavidin can be replaced by an antibiotinIgG conjugate. 160
7.2 c. Rinse. TrisHCl/NaCl buffer Indirect reaction protocol a. Block nonspecic sites. Blocking buffer
A change of buffer is sometimes advisable. 20 min An inhibition step for endogenous biotins may be carried out immediately before incubation with streptavidin. The dilution depends essentially on the label used. This balances the osmolarity of the tissue after the posthybridization washing steps. The dilution depends essentially on the label. A change of buffer is sometimes necessary for the detection step. See Section 7.2.3.
10 min
b. Form the biotinstreptavidin complex. Streptavidin 1:50 in Tris 100 l/ HCl/NaCl buffer section 6090 min c. Rinse. TrisHCl/NaCl buffer 20 min d. Incubate the conjugated biotin Diluted to 1:20 to 1:50 in TrisHCl buffer e. Rinse. TrisHCl buffer Following step Detection
60 min
20 min
7.2.3 Revelation
Enzymes require a further step to be observed by light microscopy. 7.2.3.1 Overview Essentially alkaline phosphatase and peroxidase The activity of the enzyme catalyzes the precipitation of the chromogen as a colored substance deposited at the reaction site. The conjugated label
Enzymatic activity is revealed by stained reactions that can use different chromogens, depending on the desired color of the precipitate.
7.2.3.2
Alkaline phosphatase
The chromogens (substrates) that are most widely used with alkaline phosphatase are: NBT-BCIP Fast-Red NBT-BCIP NBT (nitroblue tetrazolium)
N C N N OCH3 N H3CO N N NO2 O2N N N C
See Appendix B6.2.1.1. These two substrates are soluble in dimethylformamide. C40H30Cl2N10O6 Mw = 817.70
+ 2HCl
Figure 7.6 The chemical formula of NBT. 161
Revelation BCIP (5-bromo-4-chloro-3-indolyl phosphate)

Cl O Br ONa+ N H O
P
C8H6NO4BrCIPxC7H9N Mw = 433.60
ONa+
Figure 7.7 The chemical formula of BCIP.
Advantages Sensitivity Compatibility with multiple labeling Disadvantages The reaction sometimes takes a long time. The precipitate is soluble in alcohol. Protocol a. Reagents/solutions Dimethylformamide 10X levamisole Substrates NBT BCIP TrisHCl/NaCl/MgCl2 buffer; pH 9.5 b. The different steps: Put the substrate in place 1:250 NBT-BCIP 100 l/ section Incubate under visual surveil15 min lance until the desired reaction to 24 h is obtained. Darkness Stop the reaction in distilled 5 min water. Mount in an aqueous medium. Fast-Red (CH3)2NOCH C11H12N2S (see Appendix B6.2.1.2) Used at 1 mM The detection process can take several hours. If the labeling is intense, rapid dehydration is possible (diminution of the background). Due to the formation of two precipitates
See Appendix B3.7.6. For preparation, see Appendix B6.2.2.1. It is possible to accelerate the reaction by maintaining the slides at 37C. If the degree of detection is insufcient, deposit a further 100 l of the NBT-BCIP solution. The precipitate is soluble in alcohol. The stained reaction is expressed as a chromogen that is soluble in alcohol. C7H6N3O2 (5-chloro-2-methoxy-benzenediazonium chloride [zinc chloride]) Mw = 250.90
Figure 7.8 Fast-Red formula. 162
7.2 Advantages Sensitivity Stability of the precipitate Compatibility with multiple labels Disadvantages The reaction sometimes takes a long time. The precipitate is soluble in alcohol. Reagents/solutions Dimethylformamide Fast-Red 10 levamisole Naphthol phosphate Tris/NaCl/MgCl2 buffer, pH 9.5 Protocol a. Place the ltered substrate directly on the slides extemporaneously. Fast-Red 100 l/ section b. Incubate under visual surveil15 min lance until the desired to 24 h reaction is obtained. rt, darkness c. Rinse with distilled water. d. Mount in an aqueous medium. 7.2.3.3 Peroxidase 1 min
Note: Soluble in alcohol
Detection can take up to 24 h.
(CH3)2NOCH Ready-to-use Fast-Red solutions are commercially available. 1 mM (see Appendix B6.2.1.2) See Appendix B3.7.6. For the preparation, see Appendix B6.2.1.2.
The reaction is nished when the colored precipitate is red and clearly visible. It is possible to accelerate the reaction by maintaining the slides at 37C. To stop the reaction The precipitate is soluble in alcohol. See Appendix B8.1.
The peroxidase activity (oxidation of the appropriate substrate) produces an insoluble colored sub stance (precipitate), which materializes the reaction. The chromogens (substrates) appropriate to peroxidase are: 3-Diaminobenzidine tetrachloride (DAB) 3-Amino-9-ethylcarbazole (AEC) DAB
The electron donor is hydrogen peroxide.
H 2N
NH2
(3-Diaminobenzidine tetrachloride) The substrate is oxidized in the presence of peroxidase, and produces a signal that is expressed as a yellow-brown precipitate.
H 2N
NH2
Figure 7.9 The formula of DAB.
163
Revelation Advantages Brown precipitate Precipitate insoluble in alcohols Counterstaining possible Very intense reaction Double-labeling possible Can be intensied by nickel salts Embedding in resin that is stable over time Less background noise To be carried out as a second reaction
Disadvantages Dangerous in powder form Possibility of background noise Requires hydrogen peroxide of good quality Dangerous waste Reagents/solutions Diaminobenzidine tetrahydrochloride 30% hydrogen peroxide TrisHCl/NaCl/MgCl2 buffer, pH 7.6 Protocol a. Place the substrate. 100 l/ section For the preparation, see Appendix B6.2.2.2. Optional. Add an enzyme-blocking agent (endogenous peroxidases, see Appendix B6.2.1.3) before the use of peroxidase conjugates. Color is brown. An overlong detection step will cause a generalized coloring of the tissue. Counterstaining is possible. The precipitate is insoluble in alcohol. The reaction is expressed as a red precipitate. C14H14N2 Mw = 210.30
N
Can be attenuated by preincubation with DAB, without hydrogen peroxide Hydrogen peroxide stable for only a few weeks Can be broken down by sodium hypochlorite C12H14N14HCl (DAB) 110 volumes See Appendix B3.7.6.
b. Incubate under visual surveil310 min lance until the desired reaction rt is obtained. c. Stop the reaction by dipping the slides in distilled water. d. Counterstain. e. Embed in resin. 3-Amino-9-ethylcarbazole (AEC)
NH2
CH2CH3
Figure 7.10 The formula of AEC.
164
7.3 Advantages Red, clearly visible precipitate Double labeling Possibility of counterstaining Disadvantage Toxic Reagents/solutions 100 mM acetate buffer, pH 5.2 AEC (3-amino-9-ethyl carbazole) Dimethylformamide 30% hydrogen peroxide
Autoradiography
The solvent is harmful if inhaled.
Soluble in dimethylformamide (CH3)2NOCH 110 volumes
Protocol a. Place the substrate. 100 l/section b. Incubate under visual surveil10 min lance until the desired reaction Darkness is obtained. c. Stop the reaction with distilled water. d. Embed in an aqueous medium. Following step Observation Oxidized AEC forms a pink-red precipitate; reaction to be checked by microscope. The precipitate is soluble in alcohol (see Appendix B8.1). See Chapter 11.
7.3 AUTORADIOGRAPHY
The lack of precision of the signal, the risk of contamination of the equipment, and the indispensable precautions involved in the use of radioactive isotopes mean that autoradiography is seldom used in in situ PCR or RT/PCR. A radioactive hybrid emits radiation, which is conventionally recorded by: Autoradiography, i.e., by a photographic emulsion that visualizes it, or Phosphoimagery. Nonetheless, this is an approach that can be used to quantify amplication by measuring the levels of gray in an autoradiogram.
For macroautoradiography, see Section 7.3.3. For microautoradiography, see Section 7.3.4. This provides direct quantication but lower resolution.
7.3.1 Principles of Autoradiography

The purpose of autoradiography is to visualize radiation or particles emitted by radioactive isotopes through their materialization as grains of metallic silver. 165
Revelation x Slide
3 2 1
y Emission of radioactive particles z Detection system Figure 7.11 Principle of autoradiography.
7.3.2 Characteristics of Emulsions

7.3.2.1 Radiation Only these isotopes, along with radiation, are recorded by the emulsion.
The isotopes most commonly used in this 35 33 method are S and P. 7.3.2.2 Photographic emulsions
These are composed of silver bromide diluted in gelatin. They either take the form of a liquid in a gel (emulsion) or a solid (lm). They are characterized by: The size of the grains The thickness of the layer The type of medium Crucial for resolution Modies the sensitivity and the resolution Distinction between macroautoradiography and microautoradiography
7.3.2.3
Exposure The intensity of the autoradiographic signal depends, rst and foremost, on the relationship between the exposure time and the half-life of the isotope. The use of autoradiographic lm, like that of a phosphoimager, makes it possible to determine the exposure time without damaging the sections.
The sample, covered by a lm or a thin layer of photographic emulsion, is stored in darkness for a period of between a few hours and several months. Exposure time depends on the radioisotope, the specic activity of the probe, and the abundance of the hybrids (and thus the number of sequences looked for in the cell). 7.3.2.4 Development
The detection process changes the latent images (see Figure 7.11) resulting from the activation of the silver salts into visible metallic silver due to the radiation.
Br + radiation Br + e + Ag + e Ag
Two chemical steps: Development, with the changing of the silver salts into metallic silver by the action of radiation. Fixation, i.e., the dissolution of the remaining salts of silver bromide.
166
7.3 7.3.2.5 Efciency
Autoradiography
The efciency of autoradiography has not been improved for some years; it remains an extremely sensitive method. 15% efciency means that it takes six disintegrations of the radioisotope to produce 1 grain of silver.
An efciency of the order of 15% is generally taken to be acceptable. Such a low level is explained by the following facts: The radiation from the radioactive isotope is emitted in three dimensions, whereas the emulsion is present only on one side of the section. The emulsion is not 100% efcient. The detection system is not perfect. Background is present. The isotopes used ( S and degree of efciency. 7.3.2.6 Resolution
35 33
This is due to innumerable factors inherent in the technique.
P) give the same
The distance between the source of the radiation and the silver grains depends on: The energy of the radiation The size of the grains of emulsion The thickness of the layer of emulsion 7.3.2.7 Artifacts
This parameter is the result of a statistical 35 33 analysis. The isotopes used (i.e., S and P) give similar levels of resolution.
Artifactual labeling (background) can have a number of potential origins: The age of the emulsion Irregularities in the sections (striations, ssures, etc.) Variations in the thickness of the layer of emulsion Traces of or radiation The presence of chemicals Excessive prolongation of the detection step Accidental causes Quantication
Some observed grains correspond to nonspecic labeling, which therefore has to be quantied and subjected to a statistical analysis. This can give rise to shadows on the sections. These can cause variations in the thickness of the emulsion layer, and thus accumulations of grains. These can occur during the dipping or drying steps, and can also result from accumulations of grains. External radiation Chemography The detection of latent pseudo-images An example is inappropriate light.
7.3.2.8
The radioactivity is quantied by measuring: The optical density of macroautoradiographs, or The number of silver grains per unit area of microautoradiographs At the tissue level A rapid, standardized method At the cell level A difcult method
167
Revelation
7.3.3 Macro-Autoradiography
7.3.3.1 Overview The lm is industrially manufactured, which guarantees homogeneity and reproducibility, and thus the possibility of making comparisons between signals.
Macro-autoradiography is used to visualize tissue or organs by apposition of a lm within the totality of a section.
x Radiation The hybrid contains radioactive isotopes ( ), which emit radiation characteristic of the element in question. Autoradiography records these emissions by means of a photographic emulsion. A = lm support B = photographic emulsion C = tissue or cells D = slide
y Exposure The emulsion placed in contact with the hybrids records the radiation emitted in the course of the exposure, in the form of latent images ().
z Development These images are turned into visible grains of silver in the course of the photographic development process. Numerous parameters are involved in the interpretation of the results.
Figure 7.12 Macro-autoradiography.
168
7.3 7.3.3.2 Autoradiographic lm Resolution Handling difculties Proportional to the cost
Autoradiography
This varies according to: The size of the grains The thickness of the lm The sensitivity There are different types of autoradiographic lm, each with its particular characteristics. Single-coated lm Advantages Good resolution Quantication possible Disadvantage Generally expensive Double-coated lm Advantages Higher sensitivity Rapid response Possible comparison of several reactions Quantication Lower cost Disadvantages Macroscopic localization, medium degree of resolution Artifacts Larger emission cone Shorter exposure time than for singlecoated lm To the test of evaluation of the signal (i.e., the visualization of a possible signal) Relative quantitative analysis Measurement of the homogeneous signal density, using single-coated lm Which means that it can be used as a control for exposure time Diminution of the dispersion of the radiation. The radioactivity can be quantied by measuring the optical density of the autoradiograph.
The choice of lm depends on the isotope used, the density of the hybrid, and the required sensitivity.
7.3.3.3 7.3.3.3.1
Protocol EQUIPMENT/SOLUTIONS Regular checks should be carried out to make sure that the box is light-tight. No sterility precautions are necessary. The size of the lm depends on the number of slides to be exposed.
Equipment Cassette the right size for the lm Tray the right size for the lm Autoradiographic lm
169
Revelation Single-coated lm Double-coated lm Developer Hyperlm H, Bio Max MR, etc. X AR 500, etc. The manufacturers instructions for the use of the lm and developer must be respected if the signal is to be optimized. Sodium thiosulfate is the basis of all xatives.
3
Fixative Solutions Developer Fixative
Dilute according to the manufacturers instructions. Use 30% sodium thiosulfate, or a reagent diluted according to the manufacturers instructions. The characteristics of the xative are less crucial than those of the developer. All the following steps must be carried out in a darkroom, with a safe light. Any trace of humidity will cause an autoradiographic chemoreaction, and thus background noise. Double-coated lm can serve as a test for the exposure time. This is very sensitive; used for quantication purposes. Exposure time is to be determined. Following the manufacturers instructions Previously cooled If this duration is exceeded, the sections may be damaged. The minimum is 1 min. To avoid any trace of lime Temperature <50C, so as not to damage the lm Presence of a standard range often useful in determining the state of the lm Densitometry
7.3.3.3.2
THE DIFFERENT STEPS
a. Place the completely dry sections in the autoradiographic box, attaching them to the bottom of the box. b. Place the autoradiographic lm. c. Expose. For double-coated lm 2448 h For single-coated lm Several days
d. Store the boxes at room temperature. e. Detect. Develop 4 min 17C Rinse in running water 1 min f. Fix. Fixative 5 min g. Rinse. Running water Distilled water h. Dry. i. Evaluate the signal. j. Quantify the signal.
30 min 1 min rt, or 37C
170
7.3
Autoradiography
7.3.4 Micro-Autoradiography
7.3.4.1 Overview x Photographic emulsion Micro-autoradiography is used to visualize the silver grains individually, and to match them to cellular structures. The photographic emulsion is applied directly to the cells, in liquid form. E = photographic emulsion C = cells S = slide y Exposure This produces latent images of the radiation emitted during exposure.
z Detection Transformation of the latent images into visible grains of metallic silver in the course of the photographic development process. Numerous parameters are involved in the interpretation of the results. Figure 7.13 Micro-autoradiography. 7.3.4.2 Types of emulsion The size of the silver bromide grains differs between emulsions. High sensitivity Average sensitivity Average sensitivity
The choice of nuclear emulsion is determined by the desired sensitivity. Size of the AgBr grains: Ilford K5 Amersham LM1 Kodak NTB2 7.3.4.3 Advantages/disadvantages
0.2 m 0.25 m 0.26 m
Advantages Cellular resolution According to the isotope used, it is possible to identify different compartments in the cell. This is not the case with macroautoradiography. 171
Revelation Quantication on a single section The thickness of the layer of emulsion must be controlled. This is done by counting the silver grains on a section. Without any particular precaution being taken, and depending on the state of the section, the surface of the emulsion is generally smooth. It also varies according to the isotope used 33 35 ( P > S ). There are variations in the thickness of the layer of emulsion from one section to another. This is essentially due to differences in the thickness and homogeneity of the layer of emulsion. Only relative values can be compared.
Disadvantages The thickness of the section is difcult to control.
Contamination is proportional to the number of slides dipped. There are difculties with repeatability. Quantication is difcult between sections.
7.3.4.4 7.3.4.4.1
Protocol EQUIPMENT/REAGENTS/SOLUTIONS All the following steps are carried out in a darkroom, with a safe light placed 1.50 m above the working surface, and humidity of 20 to 40%. Clean and light-tight To dilute the emulsion Light-tightness of the storage boxes Bubble test; storage of the desiccant For wiping the backs of the slides Autoradiographic room Always the same one, so that the slides are always at the same angle Nonsterile To liquefy the autoradiographic emulsion Ordinary Silica gel wrapped in lter paper See Appendix B8.2. From Amersham, Ilford, or Kodak Or another solvent of the embedding resin
Equipment Darkroom
Autoradiographic boxes Beaker/Joly tube/emulsion tube Black tape Clean histological slides Filter paper Safe light Slide-holder
Staining trays Water bath with the thermostat set to 43C Reagents Alcohol 100 Desiccant Nonaqueous embedding medium Nuclear emulsion Xylene Solutions D 19 developer solution Distilled water 30% sodium thiosulfate Stains: 172
See Appendix B6.1.1. Nonsterile See Appendix B6.1.2.
7.3 Methyl green, sodium acetate buffer; acetic acid, pH 4.2 0.02% Toluidine blue in water See Appendix B7.1.4
Autoradiography
See Appendix B7.1.6 (stock solution), can be diluted in oxalic acid neutralized by ammonia
7.3.4.4.2 THE DIFFERENT STEPS After dehydration and drying, the sections are dipped in the emulsion. Dilution of the emulsion a. Make two marks on a beaker, or a specially designed recipient, one to correspond to the volume of water necessary for the dilution, and the other to correspond to the total volume (water + emulsion). b. Fill with water to the lower mark. c. Leave the beaker and the emulsion at 43C for 5 to 10 min before dilution. d. Add the emulsion with a porcelain spoon, or else pour it into the beaker until it reaches the upper mark. e. Mix carefully, and shake every 20 min. Liquefaction
The manufacturers recommended dilution must be adhered to. The marks must be visible under the safe light. The temperature-regulated water bath should be equipped with beaker racks. This will liquefy the emulsion. It is possible to dilute all the emulsion, and to store it in this form in darkness at 4C. Mix slowly (to avoid producing bubbles), using a glass rod or strip. Do not use metal instruments. The emulsion must be perfectly homogeneous. Check the temperature of the emulsion, which must be constant at 43C. Too high a temperature will denature the emulsion and cause background. Eliminate all the bubbles. Avoid ripples, because the thickness of the lm must be uniform. Leave one slide without a section, for background noise control. The vertical position ensures the homogeneity of the layer of emulsion. If the tissue is damp, this will act on the crystals of silver bromide (AgBr), resulting in background. A dehydrating agent of the silica gel type should be used to absorb moisture. Store at 4C to limit the development of bacteria in the emulsion. 173
1h 43C
Coating a. Dip and remove some clean slides to clean the surface of the emulsion and eliminate air bubbles. b. Slowly dip the slides vertically in the emulsion. Remove them with a slow, uniform movement, and drain the excess emulsion on the rim of the beaker for a few seconds, then on a lter paper. Drying Dry in a vertical position. 2 hovernight rt The emulsion must be dry on the slide before storage in the autoradiographic box. Storage Store the slides in hermetic boxes containing a dehydrating agent wrapped in aluminium foil, or placed in a black bag, at 4C.
Revelation Exposure time 13 weeks Sets of slides with different exposure times can be developed together. It is difcult to calculate with precision, so detection should be carried out at intervals of several days, e.g., the rst after 7 days, and the others at further intervals of 7 days. The detection step is carried out in conditions identical to those for the coating of the slides, with a safe light. There is a problem of condensation. The box must be dry before it is opened. The temperature can be increased if the labeling is weak. The temperature can be lowered if there is a lot of background, which causes a diminution of the signal. The developer can be diluted to reduce the intensity of the signal. Rinse rapidly.
Detection
a. Take the box out at least 2 h before opening it at the temperature of the laboratory. b. Place the slides in a staining tray, and transfer them successively to the following baths: Developer (D19) 4 min 17C
Distilled water 30 s Fixation Transfer the slides successively to the following baths. 30% sodium thiosulfate 5 min Running water 30 min
With delicate tissue, the time can be reduced to 1 min. Note: Do the rinsing at the same temperature as the detection (17 to 18C). Avoid temperature differences, which could cause folds in the sections. Rinse abundantly. This is a cytoplasmic and nuclear stain. Although not recommended, a slight counterstain might be performed. The nucleus is purplish-red and the cytoplasm blue. It is always possible to reduce the thickness of the gelatin layer in an acid bath. With shaking
Distilled water 1 min Counterstaining Incubate in one of the following solutions: 0.02% Toluidine blue in water 110 min rt Methyl green, sodium acetate 15 min buffer; acetic acid, pH 4.2 rt Rinse in distilled water 3 5 min Embedding a. Dehydrate. Alcohol 70, 90, 100 Xylene
21 min/bath 2 2 min
174
7.3 b. Embed Place a drop of the medium on the section. Put a coverslip in place immediately. Following steps Observation Dark eld Bright eld Epipolarization Quantication
Autoradiography
Use permanent medium (e.g., Permount, Eukitt, Depex). Eliminate bubbles. Without staining With staining With staining
175
Chapter 8
Electron Microscopy
Contents
CONTENTS
8.1 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1.1 Objective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1.2 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . 8.1.3 Pros and Cons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1.4 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1.4.1 DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1.4.2 RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 2 3
183 183 183 184 184 185 185 185 185 186 187 188 188 189 189 190 191 191 192 193 193 195 195 195 195 195 196 196 196 197 197 197 198 199 199 199 199 179
8.2 Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.1 Pre-Embedding Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.1.1 Diagram of the Different Steps . . . . . . . . . . . . . . . 8.2.1.2 The Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 8.2.1.3 Advantages/Disadvantages . . . . . . . . . . . . . . . . . . Post-Embedding Method . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.2.1 Diagram of the Different Steps . . . . . . . . . . . . . . . 8.2.2.2 The Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 8.2.2.3 Advantages/Disadvantages . . . . . . . . . . . . . . . . . . Non-Embedding Method . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.3.1 Diagram of the Different Steps . . . . . . . . . . . . . . . 8.2.3.2 The Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 8.2.3.3 Advantages/Disadvantages . . . . . . . . . . . . . . . . . . Choice of Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.2.2
8.2.3
8.2.4
8.3 Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.1 8.3.2 Origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sampling Conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.2.1 General Precautions . . . . . . . . . . . . . . . . . . . . . . . . 8.3.2.2 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.2.3 Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.4 Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4.1 8.4.2 8.4.3 Fixative . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dilution Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4.3.1 Cells in Suspension . . . . . . . . . . . . . . . . . . . . . . . . 8.4.3.2 Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.5 Cutting Sections on a Vibratome. . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5.1 8.5.2 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cutting Vibratome Sections: Practical Details . . . . . . . . . . . 8.5.2.1 Thickness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Electron Microscopy 8.5.2.2 Speed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5.2.3 Oscillation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5.2.4 Adjusting the Apparatus. . . . . . . . . . . . . . . . . . . . . Equipment/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5.3.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5.3.2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Storage of Vibratome Sections . . . . . . . . . . . . . . . . . . . . . . . 200 200 200 200 200 200 201 202 202 203 203 203 203 204 204 205 205 205 206 206 206 207 207 207 207 209 209 209 209 209 209 211 211 213 213 213 213 213 214 214 215 216 216 217 217 217
8.5.3
8.5.4 8.5.5
8.6 Pretreatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6.1 8.6.2 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . Equipment/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6.2.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6.2.2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Permeabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6.3.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6.3.2 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Deproteinization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6.4.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6.4.2 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Treatment with DNase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6.5.1 Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6.5.2 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Postxation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.6.3
8.6.4
8.6.5
8.6.6
8.7 Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.7.1 8.7.2 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Equipment/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.7.2.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.7.2.2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.7.3.1 The Reaction Mixture . . . . . . . . . . . . . . . . . . . . . . 8.7.3.2 The Different Steps . . . . . . . . . . . . . . . . . . . . . . . .
8.7.3
8.8 PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.8.1 8.8.2 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Equipment /Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.8.2.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.8.2.2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.8.3.1 The Reaction Mixture . . . . . . . . . . . . . . . . . . . . . . 8.8.3.2 The Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 8.8.3.2.1 The Hot Start . . . . . . . . . . . . . . . . . . . . 8.8.3.2.2 The Amplication Cycles. . . . . . . . . . .
8.8.3
8.9 Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.9.1 8.9.2 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Equipment/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.9.2.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.9.2.2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
180
Contents 8.9.3 Protocol for Thick Sections . . . . . . . . . . . . . . . . . . . . . . . . . 8.9.3.1 The Reaction Mixture . . . . . . . . . . . . . . . . . . . . . . 8.9.3.2 The Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 218 218 218 220 220 221 221 221 222 222 223 224 225 225 225 225 226 226 226 227 227 228 228 228 228 228 228 229 229 230 230 230 230 231 231 231 231 231 231 231 232 232 232 181
8.10
Immunocytochemical Detection on Thick Sections. . . . . . . . . . . . 8.10.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.10.2 Equipment/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.10.2.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.10.2.2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.10.3 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.10.3.1 Direct Reaction . . . . . . . . . . . . . . . . . . . . . . . . 8.10.3.2 Indirect Reaction . . . . . . . . . . . . . . . . . . . . . . . 8.10.3.3 Epoxy Resin Embedding . . . . . . . . . . . . . . . . .
8.11
Hydrophilic Resin Embedding . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.11.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.11.2 Equipment/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.11.2.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.11.2.2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.11.3 The Different Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.11.4 Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.12
Hybridization on Ultrathin Sections. . . . . . . . . . . . . . . . . . . . . . . . 8.12.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.12.2 Equipment/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.12.2.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.12.2.2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.12.3 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.12.3.1 The Reaction Medium . . . . . . . . . . . . . . . . . . . 8.12.3.2 The Different Steps . . . . . . . . . . . . . . . . . . . . .
8.13
Immunocytological Detection on Ultrathin Sections . . . . . . . . . . . 8.13.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.13.2 Equipment/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.13.2.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.13.2.2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.13. 3 The Different Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.14 Staining. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.14.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.14.2 Equipment/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.14.2.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.14.2.2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.14.3 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.14.3.1 Epoxy-Resin-Embedded Tissue Sections . . . . 8.14.3.2 Hydrophilic Resin-Embedded Tissue Sections . . . . . . . . . . . . . . . . . . . . . . . . 8.14.4 Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.15 Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.1
Overview
8.1 OVERVIEW
8.1.1 Objective
The objective is to detect nucleic acids that are very weakly expressed, at a subcellular level of resolution, by identifying the cytological characteristics of the cells in which they are present. This method combines the advantages of in situ PCR/RT-PCR and those of electron microscopy. It is necessary, to begin with, to make sure that the nucleic acid being looked for is not detectable simply by in situ hybridization, using electron microscopy.
183
Electron Microscopy
8.1.3
Pros and Cons

Resolution is improved by three orders of magnitude, from the m level to the nm level. The identication of cells in heterogeneous tissue The characterization of physiological and/or pathological states The identication of the cell compartment containing the target The study of intracell movements As described in Chapters 1 through 7, these methods involve: Preserving target nucleic acids in situ Making target nucleic acids accessible to the tools (primers, probes, enzymes, etc.), Maintaining the amplied product in situ Visualizing the signal Checking the signal An electron microscope, an ultramicrotome, and a suitable environment are indispensable. This requires Optimal preservation of cell structure Ultrathin sections a few tenths of a nm thick Compatibility of the observation method with the contrast and the signal The more nely tuned and sophisticated the technique, the more indispensable the checking procedures (see Chapter 9).
Visualization at the electron-microscopic level has undeniable advantages: Visualization of cell architecture and the different organelles
Subcellular localization of the target nucleic acid But there are also drawbacks, namely: Those inherent in in situ RT-PCR methods
Drawbacks linked to ultrastructural analysis: Logistics Methodology
Conrmation of the results
8.1.4 Applications
This ultrastructural approach applies equally to the detection of: DNA RNA as well as to the demonstration of their absence. The ultrastructural in situ PCR method The ultrastructural in situ RT-PCR method Absence of contamination or transmission of a nucleic acid support
184
8.2 8.1.4.1 DNA
Methods
The target DNA must be exogenous to the host cell so that the cells in which it is present can be picked out among a cell population. This DNA can come from: Viruses Fungi Bacteria, yeasts Transfection products
Except for the genomic DNA of the host cells Latent contamination; incorporation into the genome Contamination, symbiosis, etc. Contamination, symbiosis, etc. Studies of kinetics, effectiveness, or sexual transmission
8.1.4.2
RNA Measures must be taken to prevent RNase action. The most common Or the expression of a DNA virus Or DNA expression Or DNA expression The effectiveness of transfection
RNA is present in only a small number of copies, which must therefore be carefully preserved. It can have different origins: Genomic expression Viruses Fungi Bacteria The expression of a transfection product
1 2 3
8.2 METHODS
In electron microscopy there are three main methods for detecting nucleic acids and proteins, and thus three corresponding approaches to in situ PCR/RT-PCR: A non-embedding method A post-embedding method This uses frozen tissue sections or cell fractions (e.g., chromosomes). The tissue or cell is rst embedded in a resin (usually hydrophilic). The amplication reaction is carried out on ultrathin sections. The amplication reaction is carried out on thick sections which are then embedded in resin (epoxy or hydrophilic) before cutting ultrathin sections.
A pre-embedding method
8.2.1 Pre-Embedding Method

Here, as the name indicates, PCR/RT-PCR is carried out on thick sections, which are subsequently embedded in resin to make ultrathin sections for the detection of the amplied product, and for observation. 50 to 200 m 60 to 100 nm
185
Electron Microscopy 8.2.1.1 Diagram of the different steps
186
8.2 8.2.1.2 The different steps
Methods
The possibilities of stopping the reaction are very limited in this protocol. Paraformaldehyde is the most widely used xative.
Fixation This step requires that particular precautions be taken (xation by perfusion, then xation by immersion) to obtain a tissue or cell structure that is homogeneous throughout the thickness of the sample. Thick sections These are made with a vibratome. They can be from 50 to 100 m thick. Pretreatments The aim here is twofold: To make the tissue permeable to all the reagents To make the target nuclear acid accessible to the reagents PCR/RT-PCR This step is carried out on oating sections in a PCR tube. The reaction takes place in thick sections by incubation in the reaction medium. In theory, it is possible to carry out: Direct amplication
pected.
RNase-free conditions must be res-
Permeabilization Deproteinization The idea is to achieve a compromise between sensitivity and the preservation of cell structure. The tube is placed in a thermocycler, which follows a predened program of cycles. The incorporation of labeled nucleotides during the synthesis of amplied products (see Chapter 4) The detection of amplied products by an additional hybridization step (see Chapter 4)
Indirect amplication In practice, the difculty of eliminating labeled nucleotides means that the direct method is lacking in specicity, and that the indirect method is strongly recommended. Washing This prevents the diffusion of amplied products. Hybridization This is carried out on thick sections before embedding. Washing This eliminates nonspecic hybrids. Detection By an indirect immunocytological reaction: a. If it is carried out before the embedding step, the antigenic label is detected by an enzyme.
This step is crucial. It requires a negative internal control (see Chapter 9). It has been shown, in ultastructural in situ hybridization, that the penetration of the probe in the tissue is not a limiting factor. This is a step that needs to be optimized. Its effectiveness must be checked. For the detection of a hybrid bearing an antigenic label Generally peroxidase
187
Electron Microscopy b. If it comes after the embedding step, the antigenic label is detected by a colloidal gold conjugate. Embedding In epoxy resin if the detection step comes before the embedding step. In hydrophilic resin if the detection step comes after the embedding step and ultrathin sections are used. Contrast and observation After a more or less intense contrast, according to the chosen protocol, the sections are observed in the classical way by transmission-electron microscope.
In this case the sections are easy to make. The indirect immunocytological reaction is carried out by oating ultrathin sections on drops of the different antibodies. Diaminobenzidine (DAB), a peroxidase substrate, is made opaque to electrons by contrasting with osmium tetroxide. See Section 8.10.2.2.
8.2.1.3
Advantages/disadvantages
This currently seems like the most realistic method. Advantages Conservation of amplied products Good morphological preservation, which facilitates the identication of subcellular structures Utilization of a thermocycler with tubes Possibility of multiple labeling Within thick sections Similar to that obtained with ultrastructural in situ hybridization. It should, however, be noted that in this case the nuclear structures are denitively destroyed. The handling of oating sections in PCR tubes presents no difculties. If the detection is carried out after the embedding step. It is a delicate operation, or even impossible in the other case. Storage is not possible without compromising the experimental results. It requires several days. The signal diminishes as one advances further into the section.
Disadvantages The operation cannot be stopped before the embedding step. The method takes a long time. The embedding has to be done at, and with great care, to facilitate the recovery of the rst few sections.
8.2.2 Post-Embedding Method

This is a classical method in electron microscopy, where the amplication reaction is carried out on ultrathin sections of resin-embedded tissue. At present, only hydrophilic resins give satisfactory results.
188
8.2 8.2.2.1 Diagram of the different steps
Methods
8.2.2.2
The different steps
This method can be used with most hydrophilic-resin-embedded samples intended for immunocytological studies. Fixation by perfusion also gives excellent results.
Fixation The most commonly used xative is paraformaldehyde. In standard conditions, xation is carried out by immersion.
189
Electron Microscopy Embedding A hydrophilic resin is used cold after partial dehydration of the tissue. Polymerization is possible with ultraviolet radiation or heat. Making ultrathin sections Their thinness is no advantage in terms of the amplication reaction, which in this case is a surface reaction. Pretreatment Deproteinization is necessary. PCR/RT-PCR Indirect amplication is used on ultrathin sections. Washing This is not very important, and of low stringency. Detection Following hybridization with an antigenic labeled probe, an indirect immunocytological reaction is the most common. Colloidal gold is the label of choice. Contrast Neutral uranyl acetate is the most commonly used. The time required is 30 or 40 min longer for hydrophilic resins than for epoxy resins. Observation This uses transmission-electron microscope at low voltage.
Examples are Lowicryl or LR White. According to the type of resin used. There is little difference between these two types of polymerization. Neither facilitates the amplication reaction. In general, around 80 to 100 nm
Without this, no amplication will take place. A direct reaction is possible if all the necessary checks are carried out. Because amplication is exclusively a surface reaction in these conditions, the amplied products can be removed very easily. A direct immunocytological reaction is also possible, but it is a little less sensitive. Contrastanting substances with a high pH, such as lead citrate, should be avoided, as they denature the hybrids. See Chapter 11. When sections have been weakened by temperature variations over the course of the cycle, high voltages and currents can lead to tearing.
8.2.2.3
Advantages/disadvantages Provided that the tissue was obtained and xed in suitable conditions Not greatly altered by the amplication reaction A detection method using colloidal gold particles
Advantages Can be used to carry out retrospective studies of embedded samples A high degree of morphological preservation High resolution Rapidity Disadvantages The accessibility of the material to be amplied is limited to the surface of the sections. Amplied products diffuse into the reaction medium. For observation purposes, the sections are delicate. 190 There is little amplication. These products do not remain on the surface of the section. This is mostly due to the properties of the hydrophilic resin.
8.2 The practical side of the amplication procedure.
Methods
The way the grid is kept in place depends on the experimenters talent for improvisation.
8.2.3 Non-Embedding Method

The in situ PCR/RT-PCR reaction is carried out with non-embedded tissue sections, in general with frozen sections or cell fractions placed directly on the grid of the electron microscope. 8.2.3.1 Diagram of the different steps
Ultrathin sections are used.
191
Electron Microscopy 8.2.3.2 The different steps
Fixation As with the two previous methods, paraformaldehyde is the most commonly used xative. Cryoprotection The aim here is to limit the morphological consequences of the freezing process. The cell water is replaced by a water/cryoprotectant mixture so that the freezing can take place with a minimum of ice crystal formation. Freezing The aim here is to harden soft tissue in preparation for the production of ultrathin sections. Making ultrathin sections These are made by cryoultramicrotomy. They are ~100 nm thick. Pretreatments With this non-embedding method, using ultrathin sections, the proteins associated with the nucleic acids are the only limiting factor. PCR/RT-PCR This is carried out by direct incubation of the ultrathin sections placed on an electron microscope grid, on a drop of the reaction mixture. Washing To eliminate amplied products which are not linked to the tissue section, and which have diffused, washing steps are carried out on ultrathin sections by direct contact with the washing buffer. Detection The amplied product is detected directly on the sections by an indirect immunocytological reaction. The most commonly used label is colloidal gold.
An indispensable step The cryoprotectant should interefere neither with the nucleic acids nor with the cell or tissue structures. This is the most complex step, but also the one on which the preservation of ultrastructure depends. Utilization of a cryoultramicrotome
A small amount of deproteinization is sometimes necessary. The absence of an embedding structure is, paradoxically, a limiting factor for this method. After ve amplication cycles, the tissue is, if not destroyed, at least unrecognizable. This treatment is brief for the same reasons as before, and the absence of an embedding medium facilitates the accessibility of the products. On the other hand, there is a high risk of eliminating all the amplied products. It is also possible to carry out a direct immunocytological reaction. It gives a better degree of resolution than DAB.
192
8.2 Contrast and embedding After contrasting with aqueous uranyl acetate, ultrathin frozen tissue sections are generally embedded in a medium that will limit their desiccation. The fact is that numerous artifacts are due to the loss of the water contained in the sections during the drying process. Observation 8.2.3.3 Advantages/disadvantages
Methods
Methylcellulose is the most commonly used medium. The conservation of tissue water and soluble cellular molecules gives tissue treated by this method a particular cytological appearance (grayish cytoplasm). See Chapter 11.
Advantages This is a rapid method. The resolution of the signal is good. It is possible to use tissue or cells from collections. Disadvantages The delicate nature of ultrathin frozen tissue sections limits the number of cycles that can be carried out. The loss of cellular integrity due to the hightemperature cycles is a major disadvantage. There is a high risk of loss of tissue architecture. Specic equipment is required. Resulting in a limited amount of amplication Risk of the diffusion and disappearance of amplied products during the washing step Tissue organization fundamental to cell identication A cryoultramicrotome Using colloidal gold particles If the samples are stored in liquid nitrogen
8.2.4 Choice of Method

The choice of method depends on: Objectives Feasibility Reliability of detection The following table sets out these different factors. The expected result must reply to a question. The availability of the necessary equipment and samples. According to the nature of the samples.
193
Electron Microscopy Postembedding Method Nonembedding Method
METHODS
Pre-embedding Method Detection before Embedding vibratome no Detection after Embedding vibratome no
CRITERION Special equipment Retrospective study of samples from collections Preparation of the sample Experimental conditions Degree of simplicity Duration Reverse transcription Amplication Number of cycles Degree of diffusion of amplied products Effectiveness Detection Direct reaction Indirect reaction Label Resolution of the signal Morphological preservation Multiple labeling Feasibility Conclusion
no yes long
cryoultramicrotome yes extemporaneous
extemporaneous extemporaneous
average long on oating sections on oating sections >20 limited high possible possible antigenic or radioactive low good difcult good ++
average long on oating sections on oating sections >20 limited high possible yes antigenic good good yes good +++
high short on ultrathin sections on ultrathin sections >20 high low no yes antigenic good average yes average +
average short on ultrathin sections on ultrathin sections <5 high very low no yes antigenic good poor possible average
In conclusion, the pre-embedding method is the only one that gives satisfactory results at present. The choice between the two variants of this method depends on the required resolution of the signal and the sensitivity. 194
The diffusion of amplied products into the reaction medium is the major drawback with the post- and non-embedding methods. Resin-embedding limits the detection of the amplied products to the surface of the section, but it makes multiple labeling possible.
8.3
Samples
8.3 SAMPLES
8.3.1 Origin
The target nuclear acid can be present in: Cells Tissue An organism A biological sample from a cell culture (suspension or monolayer) Organ or biopsy Possible
8.3.2 Sampling Conditions

These conditions must be optimal if the small number of copies of nucleic acid available are to be conserved, along with the morphological characteristics of the tissue or cells being studied. The preservation of RNA targets is essentially a question of inhibiting, and if possible, completely preventing, RNase activity. With DNA, DNase activity is not a real problem. 8.3.2.1 General precautions Some of these precautions also apply to electron microscopy. Close-to-sterile conditions limit the risk of contamination by RNase. The sampling must be carried out rapidly to limit the risk of partial dehydration or proteolysis. 3 Of the order of mm for non- or postembedding methods. With the pre-embedding method, it can be the same as that of samples used in light 3 microscopy (1 cm ). It should be close to 4C to limit enzymatic activity (proteolytic, RNase, etc.). Whatever the level of observation (light microscopy or electron microscopy), the experimenter must always seek a compromise between the preservation of the target and that of the cytological characteristics. Gloves must be worn. The equipment used (asks, tubes, surgical instruments, etc.) must be either sterilized or single use.
In electron microscopy, such precautions are of the classical type: Sterility Rapidity Size
Temperature
8.3.2.2
Cells
Cells come from sterile environments, and so handling them does not require any particular precautions. It is enough to eliminate the biological or culture medium and carry out a wash in a buffer after cytocentrifugation.
It could interfere with the xative. The one that contains the xative. 195
Electron Microscopy 8.3.2.3 Tissue x Removal of the tissue

1
y Washing in a buffer
2 3
z Cutting into fragments 3 12 mm for postembedding methods 3 0.51 cm for vibratome sections used in the preembedding method
Fixation
Figure 8.1 Tissue sample preparation.
Following step Fixation
See Section 8.4.
8.4 FIXATION
The general principles and operating conditions are almost identical to those that apply to light microscopy. The differences are conned to the choice of xative, which is more restricted, along with that of the dilution buffer and the duration of the step. See Chapter 2.
8.4.1 Fixative
There are two reticulating xatives that are compatible with the amplication reaction: Paraformaldehyde Glutaraldehyde See Chapter 2. Usually paraformaldehyde prepared extemporaneously is used (see Appendix B4.3). The concentration must be extremely low (<0.2%) so as not to inhibit the amplication reaction. The preservation of the structures obtained is an advantage both in terms of morphological appearance and in limiting the diffusion of amplied products. For the moment, the range of compatible xatives is limited. No comparative studies have yet been carried out.
Other xatives are relatively incompatible either with the preservation of ultrastructure (e.g., formol) or with that of the nucleic acids (e.g., osmium tetroxide). 196
8.4
Fixation
8.4.2 Dilution Buffer

Its most important characteristics are: Osmolarity Saline concentration is the factor that has most inuence on the aqueous equilibrium, and thus the cell volume. This must always be close to neutrality so as not to disturb the equilibrium of the intracellular enzymes. The intrusion of RNase must absolutely be avoided. One of the classical buffers should be used, without any addition, e.g., phosphate buffer or PBS (see Appendix B3.4.3).
pH
Drawbacks with the method: Sterility Composition
8.4.3 Protocols
8.4.3.1 Cells in suspension and monolayer cell cultures
x Cells in suspension in the xative y Fixation, then centrifugation z Removal of the supernatant { Resuspension, then washing
Figure 8.2 Fixation of cells in suspension.
197
Electron Microscopy
x Fixation in the culture box y Washing
z Scraping the cells { Recovery
| Centrifugation } Removal of the supernatant
Figure 8.3 Fixation of monolayer cell cultures. a. Fix in a culture box. b. Wash in a buffer. 15 min 4C 3 5 min This is a cell monolayer a few m thick. It is better to increase the number of washes than their duration. Scrape as delicately as possible. Centrifuge to remove the supernatant.
c. Scrape the cells. d. Recover the cells in Eppendorf tubes. e. Centrifuge. <1000 g 8.4.3.2 Tissue
x Immersion of the samples in the xative y Washing in a buffer
Figure 8.4 Fixation of tissue fragments by immersion. a. Immerse the tissue fragments in the xative. b. Wash in an excess of buffer. 6090 min 4C 3 30 min 4C The length of time will vary according to the size of the samples. Use the same buffer as for the xation.
198
8.5 Following steps Production of vibratome sections Pretreatments
Cutting Sections on a Vibratome
For tissue For cells
8.5 CUTTING SECTIONS ON A VIBRATOME

8.5.1 Equipment
The vibratome is a vibrating microtome that is used to make thick sections (50 to 200 m) of xed tissue. The sections are recovered in PBS. So-called oating sections x Knife holder y Tray containing the buffer z The sample, attached to the nonorientable support block { Control panel | Magnifying glass support } Magnifying glass
Figure 8.5 Vibratome or vibrating microtome.
8.5.2 Cutting Vibratome Sections: Practical Details

The sample is attached to a holder in the tray containing the buffer. The blade vibrates, cutting the tissue as it moves forward. The quality of the section depends on three parameters: Thickness The speed at which the vibrating blade moves forward The oscillation or vibration of the blade This is determined by the vertical movement of the vibrating blade.
8.5.2.1 Thickness Expressed in micrometers, it must be 20 m and 200 m.
The structure and hardness of the tissue limit the possibilities. There are the limiting values for the use of this method. 199
Electron Microscopy 8.5.2.2 Speed
The speed at which the blade moves through the tissue will depend on the hardness and heterogeneity of the tissue.
It will be high if the tissue is hard. It will be low if the tissue is soft and/or heterogeneous.
8.5.2.3
Oscillation The frequency of vibration can be chosen to suit the particular type of tissue.
This is the vibrating movement of the blade that makes the section.
8.5.2.4
Adjusting the apparatus
The settings of the apparatus take account of three characteristics of the sample: size, hardness, and homogeneity. Size The thickness of the section rises with the size of the sample. Hardness The harder the sample, the higher the risk that a thick section will break. Thickness will therefore be inversely proportional to hardness. Homogeneity Slow oscillations reduce tissue heterogeneity, and give sections of better quality.
Thickness: Speed: Oscillation: Thickness: Speed: Oscillation: Thickness: Speed: Oscillation:
8.5.3.1 Equipment 20C, 80C A waterproof glue Sterile (e.g., a mounted needle or a stoppered Pasteur pipette) Sterile
Freezer Cyanolite Tool for recovering the sections Eppendorf tubes 8.5.3.2 Solutions
Cryoprotective agents 10% dimethylsulfoxide (DMSO) in 100 mM phosphate buffer
A good cryoprotectant, but it is also an organic solvent, which may damage membranes
200
8.5 10% glycerol in 100 mM phosphate buffer 30% saccharose in 100 mM phosphate buffer 70 ethanol 100 mM phosphate buffer
Cutting Sections on a Vibratome
Toxic, but highly penetrative Chemically the most neutral cryprotector See Appendix B3.4
8.5.4 Protocol
a. Pay attention to the positioning of the sample. The support block is not orientable, which means that the position of the sample is denitive. Use a waterproof glue (e.g., Cyanolite). Generally a phosphate buffer (see Appendix B3.4) This also allows the adjustment of the vibratome (speed and oscillation) to be checked. If a section remains attached to the sample, detach it with a sterile tool (e.g., a punch). They should be processed immediately. Storage is to be considered as a second-best option.
b. Adhere the sample (already xed) to the support block. c. Fill the tray with the buffer. d. Position the blade at such a height that it grazes the sample. e. Make a number of passes with the knife to obtain a clean cutting surface. f. Choose the thickness of the 50100 m sections. g. Float the sections in the buffer. h. After cutting, recover the sections with a sterile tool and place in Eppendorf tubes containing a buffer solution.
Figure 8.6 Thick vibratome section after xation. Following steps Storage Pretreatments See Section 8.5.5. See Section 8.6.
201
Electron Microscopy
8.5.5 Storage of Vibratome Sections

The only way the procedure can be interrupted is to freeze the sections immediately after they have been cut. Freezing protocol a. Divide up the required number of sections among Eppendorf tubes containing the cryoprotection agent. b. Freeze. 20C 80C 196C The main disadvantage of the preembedding method is the length of time needed to carry it out. 30% sucrose in phosphate buffer is the most widely used. 10% glycerol or 10% DMSO in phosphate buffer are alternatives. Very slow freezing Slow freezing Rapid freezing Whatever the mode of freezing, the tissue structure will be affected. But the amount of damage will be inversely proportional to the speed of freezing. The sections are in a liquid medium, so the risk of desiccation is low. It is best to store sections in the cryoprotection liquid. The lower the storage temperature, the longer it takes for recrystallization to occur. See Section 8.6.
c. Store.
20C A few days 80C A few months 196C Without limit
Following step Pretreatments
8.6 PRETREATMENTS
Reverse transcription and amplication reactions are difcult to carry out on ultrathin sections, which means that thick sections and isolated cells in suspension are the only types of material that can be used in PCR/RT-PCR techniques with electron microscopy. After the xation of the sample and the cutting of the sections, the tissue must be prepared in such a way as to render the target nucleic acid accessible to the different reagents for the amplication steps.
For thick sections of the preembedding method (non-embedded tissue)
Essentially by permeabilization and/or deproteinization
202
8.6
Pretreatments
8.6.2.1 Equipment Or incubator Slow The sterility of the solutions is crucial to the success of the following steps (see Appendix A1.1). See Appendix B2.19. If a precipitate appears, discard (see Appendix B2.20). Store for 1 month at room temperature after sterilization (see Appendix B3.4.1). Store at 4C or room temperature (see Appendix B3.5). Store at 4C or room temperature (see Appendix B3.7.2).
Water bath (37C) Shaker 8.6.2.2 Solutions
9 NaCl 70, 95, and 100 ethanol 10 N sodium hydroxide Buffers 10X phosphate buffer, pH 7.4 20X SSC (standard saline citrate) buffer, pH 7.0 TrisHCl/CaCl2 buffer, pH 7.6
203
Electron Microscopy Detergents 0.2% digitonin in phosphate buffer 0.2% saponin in phosphate buffer 0.2% sarcosyl in phosphate buffer 0.05% Triton X-100 in phosphate buffer Paraformaldehyde, 100 mM 4% phosphate buffer, pH 7.4 Proteases 1 mg/ml proteinase K, Tris (20 mM)/ CaCl2 (2 mM) buffer, pH 7.6 1 mg/ml pronase, 100 mM phosphate buffer, pH 7.4 Sterile water
See Appendix See Appendix See Appendix See Appendix See Appendix
B3.4. B3.4. B3.4. B3.4. B4.3. Store at 4C.
See Appendix B2.14.1. Store at 20C in 50 l aliquots. See Appendix B2.14.3. Store at 20C in 50 l aliquots. To be used only at the time of opening the bottle. Otherwise, use DEPC water (see Appendix B1.2).
8.6.3 Permeabilization
8.6.3.1 Overview This step requires a great deal of care, in that it involves a loss of cell integrity.
The different reagents must be allowed access to the target nucleic acid by partially destroying the cell membranes (see Figure 8.7).
Figure 8.7 Permeabilized thick vibratome section. The cell membranes are made up of phospholipids, which can be solubilized by reagents such as: Alcohol Detergents Enzyme Advantages A high level of sensitivity Partial deproteinization Ethanol Digitonin, saponin, or sarcosyl, Triton X-100 Lipase This produces an increase in the penetration of the different reagents. This means that the deproteinization step can be curtailed.
204
8.6 Disadvantages Ultrastructural damage An increase in the diffusion of amplied products
Pretreatments
There is some loss of cellular material. The destruction of structures, and membranes in particular, favors the diffusion of amplied products. It is often recommended that after the permeabilization step the structures should be restabilized by postxation.
8.6.3.2
Protocol This must be tested for each new batch.
a. Incubate the sections with one of the following: 0.05% Triton X-100 in 30 min phosphate buffer 0.2% saponin in phosphate 30 min buffer 0.2% sarcosyl in phosphate 30 min buffer 0.2% digitonin in phosphate 30 min buffer b. Rinse the sections in the same buffer: Phosphate buffer 3 10 min Following steps Deproteinization Reverse transcription Amplication
Do not allow to dry out. See Section 8.6.4. See Section 8.7. See Section 8.8.
8.6.4 Deproteinization
8.6.4.1 Overview This is a step that needs to be checked.
Eliminate the proteins associated with the nucleic acids to make the latter accessible to the reagents.
Figure 8.8 Deproteinized thick vibratome section.
205
Electron Microscopy This step can be carried out using: Enzymes
Detergents 8.6.4.2 Protocol
Specic and rapid Normally proteinase K, although other enzymes such as pronase or pepsin can also be used (see Chapter 2) Simultaneous permeabilization; but it is difcult to check the two reactions (see Chapter 2)
a. Incubate the sections successively in: 20 mM TrisHCl/2 mM 15 min CaCl2 buffer, pH 7.6 rt Proteinase K in 15 g/ml 1h TrisHCl/CaCl2 buffer rt or 15 min 37C b. Rinse: 100 mM phosphate buffer, 2 15 min pH 7.4
Room temperature Incubation concentrations and times depend on the delicacy of the tissue. They must be checked for all new experiments. The choice of buffer affects the activity of proteinase K (e.g., the absence of CaCl2 lowers its activity: e.g., the use of a TrisHCl/EDTA buffer). Here, the aim is to prevent proteinase K action, and to eliminate NH2 groups from the TrisHCl/CaCl2 buffer. Optional
8.6.5 Treatment with DNase

8.6.5.1 Principles
Destroys the nucleic acids present in the tissue sections by DNase I: This prevents the formation of unwanted hybrids; Control sections by destroying target nucleic acids.
Permits selection of RNA
Negative control of target nucleic acid
Figure 8.9 Thick vibratome section pretreated by DNase.
206
8.7 8.6.5.2 Protocol
Reverse Transcription
The enzyme (DNase) must be of excellent quality and not be contaminated by any other enzyme. a. Incubate the sections alternatively with: DNase diluted in 1 mg/ml TrisHCl/NaCl buffer 30 min 37C b. Wash freely in Tris HCl/NaCl buffer. 5 5 min at rt
This is also important for the destruction of nucleic acid targets for controls.
The concentration must be tested. The duration may be increased or decreased. It is possible to work at room temperature. Or phosphate buffer (see Appendix B3.4). Indispensable The enzymes cause breaks in nucleic acids. Washing is necessary to remove all nucleic acid fragments, which may hybridize with the probe.
c. Do not let dry.
8.6.6 Postxation
a. Postx: 4% PF in 100 mM phosphate buffer, pH 7.4 b. Rinse the sections: Phosphate buffer Following steps Reverse transcription PCR 5 min 3 5 min Optional step See Appendix B4.3. To eliminate the enzyme Eliminates any trace of xative Do not allow to dry out. See Section 8.7. See Section 8.8.
8.7 REVERSE TRANSCRIPTION

8.7.1 Overview
Reverse transcription turns the RNA of interest into cDNA, which is the only type of nucleic acid that can be amplied by PCR. The tools (primers, enzymes, nucleotides, etc.) are identical to those used in light microscopy. See Chapter 4. This principle is identical, whatever the level of observation: light or electron microscopy. See Chapter 4.
207
Electron Microscopy The only differences concern the protocols used with thick and oating sections. The thickness of the section will limit the penetration of the reagents, and thus the effectiveness of the reaction in depth. To overcome this disadvantage, some authors use another method consisting of placing ultrathin sections on grids which are then dipped into the reaction mixture and treated as oating sections in PCR tubes. Natif RNA:
Primer:
Enzyme:
Reverse transcription complex:
cDNA:
Figure 8.10 Reverse transcription on a thick vibratome section. 208
8.7 It should also be noted that cells in suspension can be processed for electron-microscopic examination after the RT or PCR step. See Section 4.5.2.2.
Reverse Transcription
8.7.2.1 Equipment Tube thermocycler
Thermocycler for liquid-phase PCR Vortex mixer 8.7.2.2 Solutions
0.1 M DTT 10 M anti-sense primer 10 mM dNTP 100 mM phosphate buffer 40 U/l RNasin 5X enzyme buffer 9 NaCl Buffer Reverse transcriptase Sterile water
See Appendix B2.7. See Section 4.3.1. See Section 4.3.2. See Appendix B3.4.
See Appendix B2.19. See Appendix B3. See Section 4.3.3. See Appendix B1.1.
8.7.3 Protocol
8.7.3.1 The reaction mixture Final concentration: 1X Final concentration: 10 mM Final concentration: 0.5 mM Final concentration: 1 U/l Final concentration: 1 M 47.5 l
In a sterile microtube, prepare the reaction mixture: 5X RT buffer 20 l 0.1 M DTT 10 l 10 mM dNTP 5 l 40 U/l RNasine 2.5 l 10 M anti-sense primer 10 l Sterile water To a total volume of 95 l
8.7.3.2
The different steps See Section 8.6.
Four or ve thick sections are pretreated, postxed, and placed in phosphate buffer in a PCR tube. Cells in suspension are also placed in phosphate buffer in a PCR tube.
209
Electron Microscopy
Reverse transcription a. Replace the phosphate buffer by the reaction mixture, and pipette delicately to resuspend the sections. b. Add 200 U/l reverse 5 l transcriptase. c. Incubate in a thermocycler. 1h 37C 1h 42C d. Deactivate the enzyme. 215 min 94C Washing The sections are rinsed: In 0.1 M phosphate 510 min buffer, pH 7.4 In 9 NaCl 510 min Following step PCR
Final concentration: 10 U/l The nal volume is 100 l. If reverse transcriptase is MMLV (see Section 4.3.3). If the reverse transcriptase is AMV (see Section 4.3.3). At this temperature, the enzyme is destroyed.
Depending on the size of the sample Depending on the size of the sample See Section 8.8. Since the sections cannot be stored, the PCR must be carried out immediately after the RT step.
210
8.8
PCR
8.8 PCR
8.8.1 Overview
In terms of general principles and the tools required, the amplication of a DNA sequence, whether for cells in suspension or for thick sections, is exactly the same in this case as with light microscopy. Only the subsequent steps, involving observations at the ultrastructural scale, are fundamentally different. There are two possibilities: The indirect reaction (see Figure 8.11) The direct reaction (see Figure 8.12) As for the reverse transcription step, however, it is necessary to take into account the penetration of thick sections by the reagents. See Chapter 5.
As in light microscopy, this is preferable to the direct reaction (see Chapter 4). The thickness of the sections is one possible cause of artifacts.
cDNA: Primers: Enzyme:
Figure 8.11 Indirect in situ PCR on a vibratome section.
211
Electron Microscopy
Amplication complex:
Amplied products:
Figure 8.11 (continued) Indirect in situ PCR on a vibratome section.
cDNA: Primers: Enzyme: Conjugated dNTP:
Figure 8.12 Direct in situ PCR on a vibratome section.
212
8.8 Labeled amplied products:
PCR
Figure 8.12 (continued) Direct in situ PCR on a vibratome section.
8.8.2.1 Equipment
Tube thermocycler Vortex mixer 8.8.2.2 Solutions See Section 5.3.1. PCR buffer (see Chapter 5). See Appendix B2.12. See Appendix B2.19. See Appendix B4.3.2. See Appendix B3.4.1. See Section 5.3.2. See Section 5.3.3. See Appendix B1.1.
Mixture of 10 mM dNTP 10X enzyme buffer Ethanol 100, 95, 70 25 mM MgCl2 9 NaCl 4% paraformaldehyde 0.1 M phosphate buffer 10 M sense and anti-sense primer Taq or Pfu DNA polymerase Sterile water
8.8.3 Protocol
8.8.3.1 The reaction mixture
In a sterile microtube placed in ice, prepare the reaction mixture: 10X PCR buffer 10 l 25 mM MgCl2 6 l 10 mM dNTP mixture 5 l 10 M sense primer 10 l 10 M anti-sense primer 10 l Sterile water To a nal volume of 96 l Final concentration: 1X Final concentration: 1.5 mM Final concentration: 0.2 mM Final concentration: 1 M Final concentration: 1 M 55 l 213
Electron Microscopy 8.8.3.2 The different steps See Section 5.5.2.
Before starting the amplication cycles, a hot start has to be carried out. The following washing and postxation steps are necessary to the stabilization of the amplied products and cell structures.
8.8.3.2.1 THE HOT START Unless one uses Pfu DNA polymerase, whose activity is minimal below 50C, or DNA polymerase coupled to an antibody that inhibits its activity, a hot start is required to ensure the specicity of the primer matching. a. Pipette off the phosphate buffer, replace it by the reaction mixture, and carefully resuspend the sections. b. Incubate. 5 min 82C c. Add 5 U/l Taq DNA 4 l polymerase. 214
See Section 5.3.3.2. The risk of nonspecic hybridization is signicant at low temperatures.
Depending on the volume Standard temperature Final concentration: 0.2 U/l
8.8 8.8.3.2.2 THE AMPLIFICATION CYCLES These consist of the three classical phases:
PCR
Denaturation Hybridization
Extension Programming the amplication cycles: a. Three steps of PCR cycle. Denaturation 1 min 94C
It should, however, be noted that the amplication is carried out in a tube in a larger reaction volume than for liquid-phase PCR. This volume determines how long the different phases of the cycle need to last so that the temperatures are respected. See Section 5.3. It must be effective within all the cells. See Section 5.3. It must also be thermally compatible with the set of primers, and facilitate their diffusion. See Section 5.3. It must also last long enough to allow the enzyme and the nucleotides to diffuse. The duration has to be optimized, and the absence of a signal can be due to insufcient denaturation. This temperature is considered a reference value. Poor hybridization may result in a weak signal. The hybridization temperature must be optimized according to the characteristics of the primers. Insufcient extension may result in a weak signal. This is a reference value for most DNA polymerases. Necessary Reference value The sections can be left waiting for some time. The thermocycler can be set at 4C if the sections are left waiting for some time. Too few cycles result in insufcient amplication, but with larger numbers there is a risk of destabilization of the structures, and loss of the amplied products, by diffusion. During systematic assays, the amplication signal decreases after 30 cycles. The aim is elimination of diffused products. The number (X) of washes, and their duration, should be high. Resuspend the sections after each wash.
Hybridization
90 s 4560C
Extension
90 s 72C
b. Final extension. c. Stop the reaction.
10 min 72C 1 min 30C
Number of cycles This number needs to be determined empirically. It is between 10 and 25.
Washing 0.1 M phosphate buffer
X 5 min
215
Electron Microscopy Postxation An important step for: Stabilizing tissue and cell structures Fixing the amplied products
Observation and ultrastructural analysis require good morphological quality. The fragility of cell structures due to pretreatments (e.g., permeabilization, deproteinization), and temperature variations during the amplication cycles result in the diffusion and loss of amplied products during the detection steps. The foregoing baths are completely eliminated by pipetting, and the sections are carefully resuspended. There is no possibility of storage or of leaving the sections waiting. See Section 8.9. See Section 8.10.
4% paraformaldehyde 0.1 M phosphate buffer 9 NaCl Following steps Hybridization Embedding
510 min 5 min 2 min
8.9 HYBRIDIZATION
8.9.1 Overview
With indirect in situ PCR/RT-PCR, hybridization is the means used to detect amplied products, both in electron and light microscopy. The neosynthesized DNA is double stranded, and so two specic probes, sense and anti-sense, are used. These are taken from the fragment of interest so that it alone will be detected. See Chapter 6. A single probe can be used, but this will reduce the effectiveness by half. An increase in specicity.
Denaturation of the amplied products:
216
8.9
Hybridization
Probes:
Incubation in the presence of probe
Labeled hybrids:
Formation of labeled hybrids
Figure 8.13 Hybridization of amplied product on a vibratome section.
8.9.2.1 Equipment
Incubator Shaker Vortex mixer
8.9.2.2
Solutions See Appendix B2.4. See Appendix B2.5. See Appendix B2.8.
Deionized formamide 50X Denhardts solution 10 mg/ml salmon sperm DNA
217
Electron Microscopy Labeled anti-sense probe 1.25 pmol/l for an antigen 0.25 pmol/l for a radioisotope See Chapter 6. 1.25 pmol/l for an antigen 0.25 pmol/l for a radioisotope See Chapter 6. See Appendix B2.15. See Appendix B3.5. See Appendix B1.1.
Labeled sense probe
10 mg/ml tRNA 20X SSC Sterile water
8.9.3 Protocol for Thick Sections

8.9.3.1 The reaction mixture
For the ultrathin section protocol, see Section 8.12.
In a sterile microtube placed in ice, prepare the reaction mixture: 20X SSC Deionized formamide 50X Denhardts solution 10 mg/ml tRNA 10 mg/ml salmon sperm DNA Labeled sense probe 100 l 250 l 10 l 12.5 l 12.5 l 8 l Final concentration: 4X Final concentration: 50% Final concentration: 1X Final concentration: 250 g/ml Final concentration: 250 g/ml
Labeled anti-sense probe 8 l Sterile water To a nal volume of 500 l
Final concentration: 20 pmol/ml for antigens 4 pmol/ml for radioisotope Final concentration: 20 pmol/ml 99 l
8.9.3.2
The different steps The amount of handling involved is reduced, and therefore in the risk of drying out. Here, the handling of the sections is a delicate matter, but the reaction volume necessary for the incubation of sections that are well spread on slides is lower. After the hybridization step, the cell pellet is treated in the same way as a tissue sample: it is embedded and cut into sections for immunocytochemical detection.
The classical hybridization procedure is followed, with the same temperatures and durations. The sections are incubated either in PCR tubes (for oating sections) or on glass slides (for semioating sections). With cells in suspension, hybridization is carried out in tubes.
218
8.9
Hybridization
Amplification
Washes
Semifloating section
Floating sections
Cell suspensions
Hybridization
Washes
Hybridization a. Replace the phosphate buffer in which the sections are dipped by the reaction mixture. b. Resuspend the sections.
Or spread the sections on slides Or, in the case of semioating sections, cover the sections with the reaction mixture and a coverslip to avoid evaporation The amplied product is double stranded, so it has to be denatured before hybridization. On a heating block To stabilize the DNA in single-strand form The slides are incubated in a moisture chamber containing 5X SSC to maintain the same vapor saturation between the reaction mixture and the moist environment. The hybridization temperature is specic to the probes being used. It can be increased to improve specicity. If necessary, detach the coverslips with 4X SSC. Or resuspend the sections in decreasing concentrations of SSC Necessary to optimize the stringency of the washing in terms of the results (see Chapter 6) At room temperature At room temperature At room temperature 219
c. Denature.
5 min 96C 5 min Overnight
d. Cool immediately by contact with ice. e. Incubate in the thermocycler.
40C Washing a. Remove the reaction mixture by pipetting. b. Rinse: 4X SSC 2X SSC 0.5X SSC 2 10 min 2 30 min 30 min
Electron Microscopy Following steps Either: Detection using an antigenic probe and oating sections, and Embedding; or: Embedding in hydrophilic resin, and Antigenic detection on ultrathin sections. Immediately after the RT step See Section 8.10. See Section 8.10.3.3. See Section 8.11.
See Sections 8.12 and 8.13.
8.10 IMMUNOCYTOCHEMICAL DETECTION ON THICK SECTIONS

8.10.1 Overview
The label of the hybrids present in vibratomee sections is considered an antigen. Its detection is obtained by an immunocytochemical reaction. The limitation of this detection results from the penetration of the different reagents (antibodies, labels, chromogens).
See Figure 8.14.
Antibody:
Figure 8.14 Immunocytological detection on vibratome section. Two types of immunocytochemical reaction can be carried out after hybridization and washing: Direct reaction, in which the antigen/ antibody complex is labeled. See Figures 7.3 and 7.4.
Utilization of a primary antibody: Immunoglobulins of polyclonal or monoclonal origin (IgG), or Fragments of IgG: Fab or F(ab)2, conjugated to a label
220
8.10 Indirect reaction, in which the antigen/ antibody complex formed during the rst step is not labeled. This is the second step, with the second conjugated antibody, which reveals the formation of the complex. The most widely used label for this detection method using oating sections is peroxidase, an enzyme that catalyzes the precipitation of chromogen. Advantage This reaction takes place within the thickness of the section. Disadvantage The precipitate is diffused, and often does not give the precision required for observation at the subcellular level.
Immunocytochemical Detection on Thick Sections The use of a nonconjugated primary antibody, followed by a second conjugated antibody directed against the species that was used to make the rst one
Diaminobenzidine (DAB) is the most widely used chromogen. It oxidizes into a brown precipitate, and is made opaque to electrons by osmicated postxation. Unlike colloidal gold, which, in spite of the use of small-sized particles (1 to 5 nm), is a surface label.
8.10.2.1 Equipment Horizontal slow shaker 16-well culture box Cover slide made of silicon glass Vortex mixer Solutions See Appendix 6.2.1. See Appendix B3.4.3. See Appendix B3.7.5. Other agents can be added to the blocking buffer (e.g., sh gelatin, ovalbumin, nonsweetened skimmed milk). See Appendix B3.7.5. Add 1% goat serum and 0.01% Triton X-100. See Appendix B3.4.3. See Appendix B6.2.2.2. 30% H2O2 = 110 volumes. Monoclonal or polyclonal IgG, Fab fragments Conjugated = peroxidase. 221 Sterile See Appendix A3.2.
8.10.2.2
Buffers: blocking buffers PBS buffer TrisHCl/NaCl buffer, pH 7.6 TrisHCl/NaCl/ovalbumin buffer, pH 7.6
TrisHCl/NaCl/goat serum/Triton X-100 buffer, pH 7.6. Ethanol 30, 50, 70, 80, 90, 95, 100 Fixative 1% osmium tetroxide/PBS Detection solution (DAB) Hydrogen peroxide Direct reaction antibody conjugated anti-label IgG
Electron Microscopy Indirect reaction antibody Conjugated anti-label IgG (species X) Conjugated anti-species X IgG Epon Epoxy inclusion medium Hexahydrophtalic anhydride Succinic dodecenyl anhydride Nadic methyl anhydride (dicarcoxylic norborene anhydride-2,3) 2,4,6-Tridimethylamine methyl phenol Propylene oxide
Monoclonal or polyclonal IgG, or F(ab)2, Fab IgG fragments Peroxidase Epoxy resin See Appendix B5.2 Epikote 812 DDSA (hardener) MNA (hardener) DMP 30 (accelerator)
8.10.3 Protocol
8.10.3.1 Direct reaction The sections must never dry out. To reequilibrate the tissue, after the posthybridization washings, in a buffer that is better than SSC for immunocytochemical reactions Indispensable for limiting nonspecic reactions Optional, since endogenous peroxidase can be destroyed during the different hightemperature stages of the PCR (see Appendix B6.2.1.3) Its existence can be checked by leaving out the conjugated antibody. 30% H2O2 = 110 volumes
a. Incubate thick sections in a 16-well culture box: TrisHCl/NaCl 200 l/ buffer, pH 7.6 thick section 10 min b. Block nonspecic sites: TrisHCl/NaCl 200 l/ buffer; ovalbumin, thick section pH 7.6 60 min rt c. Inhibit endogenous enzymatic activity:
3:100 in TrisHCl buffer 35 min d. Incubate in the conjugated anti-label antibody: 200 l/ Diluted to 1:100 in TrisHCl/ NaCl buffer, thick section pH 7.6 Incubate. 1 night 4C
Hydrogen peroxide for endogenous peroxidase
Formation of the antigenantibody complex. Primary antibody The dilution of the antibody is generally recommended by the manufacturer, but can be adapted to the intensity of the observed signal.
222
8.10 e. Rinse: TrisHCl/NaCl buffer, pH 7.6 f. Detect peroxidase activity: 0.025% DAB; Tris HCl buffer; 0.03% hydrogen peroxide; pH 7.6 g. Incubate under visual surveillance. h. Stop the reaction: PBS i. Postx vibratome sections: 1% osmium tetroxide PBS j. Rinse: PBS Following step Embedding in epoxy resin 8.10.3.2 Indirect reaction 530 min 30 min 3 10 min
Immunocytochemical Detection on Thick Sections
3 10 min
An important step Increase the number of washings to improve the signal/background ratio For the method of preparation, see Appendix B6.2.2.2. Color is brown. Too long a detection process leads to a fall in the signal/background ratio. Stop when the intensity and the signal/background ratio are correct. Indispensable before osmium tetroxide The DAB chromogen is made opaque to electrons by the reduction of osmium atoms. Rinse to eliminate the xative. See Section 8.10.3.3.
100 l/thick section 320 min rt
The rst steps, and the method of carrying them out, are identical to those of the direct reaction: Blockage of nonspecic sites Inhibition of endogenous enzymatic activity The changes begin in step d. d. Incubate the anti-label antibody (species X). Diluted 1:100 in Tris HCl/NaCl buffer, pH 7.6 200 l/ thick section Formation of the antigen/antibody complex Primary antibody The dilution of the antibody is generally recommended by manufacturers, but it must be optimized to improve the signal/background ratio.
Incubate
Overnight 4C Increase the number of washes to improve the signal/background ratio. Conjugated secondary antibody: IgG or fragments, diluted according to the manufacturers recommendations Dilution < that of the primary antibody Can be reduced if necessary
e. Rinse TrisHCl/NaCl 3 10 min buffer, pH 7.6 f. Incubate the conjugated anti-species-X antibody. 200 l 2h rt
Incubate
223
Electron Microscopy g. Rinse further. Buffer TrisHCl/ 3 10 min NaCl; pH 7.6 The steps in the direct reaction, i.e.: Detection of peroxidase activity, Checking the reaction, and Postxation of vibratome sections with osmium tetroxide are identical to those of the indirect reaction. Following steps Embedding in epoxy resin Embedding in hydrophilic resin
An important step Use an excess of buffer. See Section 8.10.3.1. See Section 8.10.3.1. See Section 8.10.3.1.
See Section 8.10.3.3. See Section 8.11. Only in the case of multiple labeling (i.e., the identication of a particular protein using colloidal gold after the detection of amplied products)
8.10.3.3
Epoxy resin embedding All the other epoxy resins (e.g., araldite, spurr) can be used with the classical protocols. The sections are processed on 16-well plates. All the following steps are carried out at room temperature. With each change of bath, eliminate as much of the previous liquid as possible.
This protocol for embedding in Epon resin is classical. Dehydration Ethanol 30 Ethanol 50 Ethanol 70 Ethanol 80 Ethanol 90 Ethanol 95 Ethanol 100 1530 min 1530 min 1530 min 1530 min 3 1530 min 3 1530 min 3 1530 min
Inltration a. Inltrate thick sections in solvent/resin mixtures at increasing resin concentrations: Propylene oxide + 2:1 v/v Epon 45 min Propylene oxide + 1:1 v/v Epon 45 min Propylene oxide + 1:2 v/v Epon 45 min b. Replace the substitute solution with Epon resin: First bath: Epon + DMP 30 224 12 h rt
The total dehydration of the samples is indispensable. Prepare the different inltration solutions extemporaneously. Use stoppered asks. Some types of plastic do not withstand propylene oxide. Steps are carried out at room temperature. This is a toxic mixture, to be handled under a ventilated hood.
Use a freshly prepared resin. Use an unstoppered ask, to obtain the complete evaporation of the solvent. This is a toxic mixture, which must be handled under a ventilated hood.
8.11 Second bath: Epon + DMP 30 Embedding Overnight 4C
Hydrophilic Resin Embedding
Avoid air bubbles. 300 l of resin per cavity is used in at embedding. This is a toxic mixture, which must be handled under a ventilated hood. With at embedding, the sections can be arranged in any given plane. For the preparation of the coverslip, see Appendix A3.2.
a. Spread the thick sections with a ne point: In a mold, for at embedding On a coverslip made of silicon glass, taking care to eliminate as much resin as possible each time. b. Place a capsule lled with resin vertically on the section. Polymerization Duration 48 h 60C Following steps Ultramicrotomy Observation
To facilitate removal from the molds, it is recommended that they be cooled to 4C for 15 to 30 min. Making semithin sections Making ultrathin sections
8.11 HYDROPHILIC RESIN EMBEDDING

This step can be carried out: After amplication, After hybridization, or After immunocytochemical detection
8.11.1 Overview
Vibratome sections are embedded in LR White resin or another hydrophilic resin. The protocol is classical, and comprises three steps: Dehydration, Substitution, and Polymerization followed by the making of ultrathin sections, either directly observed after contrasting or on which the detection of hybrids is carried out. Of the Lowicryl or Unicryl type
See Section 8.13.
8.11.2.1 Equipment Incubator Ultramicrotome Ultraviolet polymerization system >60C If possible, at low temperature 225
Electron Microscopy 8.11.2.2 Solutions See Appendix B5.3. See Appendix B5.3.2. This can be used instead of LR White. Its toxicity is a disadvantage, and it must be used only in a cryosubstitution apparatus of the AFS type (Automatic Freeze-substitution System). See Appendix B5.3.1.
Alcohol 50, 70, 95, 100 Hydrophilic resin LR White Lowicryl K4M

Dehydration Alcohol 50, 70, 95, 10 min 100 per bath Substitution Alcohol 100LR White 30 min (2:1 v/v) Alcohol 100LR White 30 min (1:1 v/v) Alcohol 100LR White 30 min (1:2 v/v) Polymerization The section, having been well spread on a very at surface in a drop of resin, is covered with a capsule. Then it is either: Placed in an incubator, or 2 days 60C Identical for other resins . Very similar for other resins
Polymerization takes place only in anaerobic conditions. Oxygen prevents polymerization. Polymerization is an exergonic reaction, which favors the denaturing of hybrids and/or amplied products. It should be carried out before hybridization. This can cause breaks in amplied products. It should be carried out after detection on the sections.
Subjected to ultraviolet radiation
23 days 20C
8.11.4 Sections
100-nm-thick sections are classically made on an ultramicrotome. After the immunocytochemical detection has been carried out, only the rst sections give a high level of signal. Great care must therefore be taken in preparing them. In other cases, it is preferable to leave out the rst few sections, which always have mechanical artifacts due handling. Particularly important if other processes have to be carried out on the sections, e.g., autoradiography or hybridization.
They are collected on collodionized and carbonized nickel grids.
226
8.12 Following steps Hybridization Immunocytological detection
Hybridization on Ultrathin Sections
8.12 HYBRIDIZATION ON ULTRATHIN SECTIONS

8.12.1 Overview
If this step in the detection of the indirect in situ PCR/RT-PCR is not carried out on thick sections, it is possible to carry it out on ultrathin sections. The principle is identical after the denaturing of the amplied products, and these are hybridized with two specic probes. See Chapter 5. The sections are embedded in hydrophilic resin after the PCR step.
Denatured hybrids: x x Denaturation
Probes: y y Incubation with the probes
Labeled hybrids: z z Formation of labeled hybrids Figure 8.15 Hybridization on ultrathin section. Advantages Resolution Multiple labeling Morphological preservation Limitation of problems due to the penetration of tools (i.e., probes and antibodies) during the detection process Disadvantage Loss of sensitivity Hybridization is limited to the surface of the sections. Colloidal gold particles can be used for detection (see Section 8.13). Simultaneous detection of the different amplied products is possible. Hybridization causes little or no damage to sections. See Chapter 7.
227
Electron Microscopy
8.12.2.1 Equipment
Slow shaking device Petri dish Needle-nosed pliers
8.12.2.2
Solutions See Appendix B2.5. See Appendix B2.8. See Appendix B2.4. See Appendix B2.20. 1.25 pmol/l See Chapter 6. 1.25 pmol/l See Chapter 6. See Appendix B2.15. See Appendix B3.5. See Appendix B1.1.
50X Denhardts solution 10 mg/ml salmon sperm DNA Deionized formamide 0.5 N NaOH Labeled anti-sense probe
Labeled sense probe 10 mg/ml tRNA 20X SSC Sterile water
8.12.3 Protocol
8.12.3.1 The reaction medium
For the protocol used with thick sections, see Section 8.9.3.
In a sterile microtube placed in ice, prepare the reaction mixture: 20X SSC 100 l Deionized formamide 150 l 50X Denhardts solution 20 l 10 mg/ml tRNA 12.5 l 10 mg/ml salmon sperm DNA 12.5 l Labeled sense probe 8 l Labeled anti-sense probe 8 l Sterile water To a nal volume of 500 l The different steps The ultrathin sections are incubated by oating the grid on the surface of a drop of hybridization buffer, and washed in the same way. Final concentration: 4X Final concentration: 30% Final concentration: 2X Final concentration: 250 g/ml Final concentration: 250 g/ml Final concentration to 20 pmol/ml Final concentration to 20 pmol/ml 189 l
8.12.3.2
The amplied products that are present at the surface of the sections are considered exogenous DNA. They are rst denatured, then hybridized with two probes, each of which is complementary to one of the strands of this DNA. The excess of the probes, and nonspecic hybrids, are eliminated by washings. 228
8.13 Denaturation a. Float the grids on a solution: 0.5 N NaOH b. Wash: SSC buffer Hybridization Incubate the sections on the reaction mixture. Washings
Immunocytological Detection on Ultrathin Sections
15 min rt 4X 3 5 min rt
The amplied product is double stranded, so it has to be denatured before hybridization. NaOH must be eliminated. To avoid evaporation, this should be carried out in a moisture chamber in 5X SSC. A longer duration rarely improves the intensity of the reaction. The temperature can be increased to improve specicity. If necessary, remove the coverslips with 4X SSC. The stringency of the washings may vary in line with the results (see Chapter 6). At room temperature At room temperature At room temperature No possibility of storage See Section 8.13.
3h rt
Incubate the sections successively on drops of: 4X SSC 2X SSC 0.5X SSC 2 10 min 2 30 min 30 min
Following step Immunocytological detection on ultrathin sections
8.13 IMMUNOCYTOLOGICAL DETECTION ON ULTRATHIN SECTIONS

8.13.1 Overview
The label or haptene incorporated into the hybrids formed during the hybridization step is considered as an antigen. Its detection takes place through the formation of an antigen/antibody complex in a direct or indirect immunocytological reaction, most often using colloidal gold for visualization. Identical to that of the immunocytochemical detection method used with thick sections (see Section 8.10)
Antibody:
Colloidal gold particle:
Figure 8.16 Immunocytological detection on an ultrathin section.
229
Electron Microscopy
8.13.2.1 Equipment
Needle-nosed pliers Paralm Petri dish 8.13.2.2 Solutions See Chapter 7. The size of the colloidal gold particles is generally 10 nm, but 5-nm particles can be used as a compromise between resolution and sensitivity. The most frequently used haptenes are digoxigenin, biotin, and uorescein. See Chapter 6. RNase-free conditions are not necessary. See Appendix B1.1. See Appendix B4.2. See Appendix B3. See Appendix B3.7.5. See Appendix B3.7.5. See Appendix B3.7.5; added with 1% ovalbumin and 0.1% Tween 20. See Appendix B3.5.
An anti-species antibody conjugated to colloidal gold particles
Anti-haptene antibodies
Distilled water 2.5% glutaraldehyde Buffers TrisHCl/NaCl, pH 7.4 TrisHCl/NaCl, pH 8.2 TrisHCl/NaCl/ovalbumin/Tween 20, pH 8.2 20X SSC

Indirect detection by an antibody directed against the antigenic molecule used. a. Incubate: Anti-haptene monoclonal antibody, 1h diluted to 1:50 in TrisHCl/NaCl rt buffer, pH 7.4, to which a blocking agent has been added b. Rinse: In the same buffer 2 5 min In TrisHCl/NaCl/ovalbumin/ 2 5 min Tween 20 buffer, pH 8.2 c. Incubate. Anti-mouse antibody conjugated to 1h 10-nm colloidal gold particles, diluted rt to 1:50 in the same buffer as before d. Rinse: TrisHCl/NaCl buffer, pH 8.2 2 5 min 2X SSC buffer 2 5 min
According to the label used Drops of the reagent are placed on a plastic lm of the Paralm type. The grids are incubated on drops of reagent, with the section side against the liquid. For blocking buffer, see Appendix B6.2.1. A pH of 8.2 important for the stability of the antibody conjugated to colloidal gold According to the primary antibody used Anti-species antibody directed against the species of the rst antibody
To eliminate all trace of TrisHCl buffer, which could interfere with the xation step
230
8.14 Fixation 2.5% glutaraldehyde in 2X SSC buffer Washing in 2X SSC Rapid rinsing in distilled water Following steps Staining Observation 5 min 2 5 min See Section 8.14. See Chapter 8.11.
Staining
Stabilization of the immunocytological reaction
8.14 STAINING
8.14.1 Overview
Place heavy metal salts (e.g., uranium, osmium, lead) in contact with the surface of the ultrathin section to render the compounds opaque to electrons. Figure 8.17 An ultrathin section after staining.
8.14.2.1 Equipment
Needle-nosed pliers Paralm Petri dish Wash bottle Solutions See Appendix B7.2.1.2. See Appendix B7.2.1.1. See Appendix B7.2.2. RNase-free conditions are not necessary.
8.14.2.2
Uranyl acetate: Aqueous 5% Alcoholic 2% Lead citrate Distilled water
8.14.3 Protocol
8.14.3.1 Epoxy-resin-embedded tissue sections 2% 1530 min
a. Incubate the grids on a drop of: Alcoholic uranyl acetate
The incubation time has to be controlled. 231
Electron Microscopy b. Rinse the grids in: Distilled water c. Incubate the grids on a drop of: lead citrate
2 5 min
The water must be ltered through a 0.2 m lter. The incubation time has to be controlled.
2% 25 min
d. Rinse the grids in: Distilled water
2 5 min Wash bottle jet
8.14.3.2
Hydrophilic resin-embedded tissue sections Good contrast is difcult to obtain on hydrophilic resin sections with aqueous uranyl acetate. The incubation time must be optimized. The water must be ltered through a 0.2 m lter.
a. Incubate the grids on a drop of: Aqueous uranyl acetate
5% 30 min
b. Rinse: Distilled water
2 5 min Wash bottle jet
c. Dry the grids.
8.14.4 Storage
The grids are stored in conditions free from dust and ultraviolet radiation, pending observation. Ultraviolet radiation is not used with epoxy resin-embedded sections.
8.15 OBSERVATION
The grids are observed by transmission electron microscopy at 60 or 80 kV. See Chapter 11.
232
Chapter 9
Controls and Problems
Contents
CONTENTS
9.1 Signal/Background Ratio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.1 Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.2 Pretreatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.3 Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.4 PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.5 Hybridization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.6 Washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.7 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sensitivity/Specicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.1 Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.2 Pretreatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.3 Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.4 PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.5 Hybridization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.6 Washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.7 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.8 Summary Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.1 Tools and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.2 Tissue. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.3 Pretreatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.4 Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.5 PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.6 Hybridization/Washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.7 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.8 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.9 Validation by Other Techniques . . . . . . . . . . . . . . . . . . . . . . . . 9.3.10 Summary Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . False Positives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.1 Nonspecic Incorporation of Labels . . . . . . . . . . . . . . . . . . . . . 9.4.1.1 Denition of the Problem . . . . . . . . . . . . . . . . . . . . . . 9.4.1.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.1.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.2 Repair of Cellular DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.2.1 Denition of the Problem . . . . . . . . . . . . . . . . . . . . . . 9.4.2.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.2.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.3 Nonspecic Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.3.1 Denition of the Problem . . . . . . . . . . . . . . . . . . . . . . 9.4.3.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.3.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.4 Nonspecic Hybridization of the Primers . . . . . . . . . . . . . . . . . 9.4.4.1 Denition of the Problem . . . . . . . . . . . . . . . . . . . . . . 237 237 238 239 240 241 242 242 243 243 244 245 246 248 249 249 250 256 256 257 257 258 259 259 260 260 261 262 263 264 264 264 264 264 265 265 265 265 265 265 266 266 266
9.2
9.3
9.4
235
Controls and Problems 9.4.4.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.4.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.5 Amplication of Genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . 9.4.5.1 Denition of the Problem . . . . . . . . . . . . . . . . . . . . . . 9.4.5.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.5.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.6 Diffusion of Amplied Products . . . . . . . . . . . . . . . . . . . . . . . . 9.4.6.1 Denition of the Problem . . . . . . . . . . . . . . . . . . . . . . 9.4.6.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.6.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.7 External Contamination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.7.1 Denition of the Problem . . . . . . . . . . . . . . . . . . . . . . 9.4.7.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.7.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.8 Summary Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . False Negatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.1 Destruction of Target Sequences . . . . . . . . . . . . . . . . . . . . . . . . 9.5.1.1 Denition of the Problem . . . . . . . . . . . . . . . . . . . . . . 9.5.1.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.1.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.2 Problems Related to the Fixation Process . . . . . . . . . . . . . . . . . 9.5.2.1 Denition of the Problem . . . . . . . . . . . . . . . . . . . . . . 9.5.2.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.2.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.3 Problems Related to Proteasic Digestion . . . . . . . . . . . . . . . . . 9.5.3.1 Denition of the Problem . . . . . . . . . . . . . . . . . . . . . . 9.5.3.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.3.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.4 Reverse Transcription Problems . . . . . . . . . . . . . . . . . . . . . . . . 9.5.4.1 Denition of the Problem . . . . . . . . . . . . . . . . . . . . . . 9.5.4.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.4.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.5 Amplication Problems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.5.1 Denition of the Problem . . . . . . . . . . . . . . . . . . . . . . 9.5.5.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.5.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.6 The Stringency of the Washes . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.6.1 Denition of the Problem . . . . . . . . . . . . . . . . . . . . . . 9.5.6.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.6.3 Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.7 Detection Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.7.1 Denition of the Problem . . . . . . . . . . . . . . . . . . . . . . 9.5.7.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.7.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.8 Summary Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266 266 266 267 267 267 267 268 268 268 268 268 268 268 269 269 270 270 270 270 271 271 271 271 272 272 272 272 272 273 273 273 273 273 274 274 275 275 275 275 275 275 275 276 276
9.5
236
9.1
Signal/Background Ratio
The quality of the detection method depends on the optimization of each stage in the procedure, from the sampling conditions to the revelation process itself. An interpretable result is obtained by respecting the fundamental principles and modifying certain factors. The validity of this result is underpinned by the numerous controls that have to be carried out.
See Sections 9.1 and 9.2. See Section 9.3.
9.1 SIGNAL/BACKGROUND RATIO

Result = Signal + Background
=
9.1.1 Sampling
Parameters
The observed result comprises the signal plus the background. Only the ratio of these two parameters enters into the nal analysis.
Sampling conditions
The sampling has to be carried out rapidly to ensure conservation of the nucleic acids. Everything possible must be done to protect the samples from the action of exogenous RNase and DNase. Preservation of nucleic acids Preservation of morphology An indispensable step See Chapter 2. Standard Often necessary to test the preservation of the nucleic acids by hybridization using a poly (T) probe Problems: the diffusion of amplied products and the preservation morphology A compromise between cellular preservation and the diffusion of amplied products 237
Fixation x Light microscopy y Electron microscopy z Types of xative Formaldehyde Other { Duration + ++
Controls and Problems +++ Provided that more drastic pretreatments are carried out Reduction in the diffusion of amplied products Better accessibility of the nucleic acids If the morphological preservation is poor, a risk of the diffusion of amplied products Seldom used method Standard Standard, for thick sections See Chapter 8. See Chapter 8. For the preembedding method
Freezing x Light microscopy Without xation
After xation y Electron microscopy Embedding x Light microscopy Parafn embedding y Electron microscopy Before embedding Without embedding After embedding Semithin sections Storage x Light microscopy Frozen sample Frozen sections Parafn-embedded samples Parafn-embedded sections y Electron microscopy Before embedding Without embedding After embedding
80C, or liquid nitrogen Necessary to store the slides in anhydrous conditions Virtually unlimited conservation period Not recommended; storage of blocks preferable Impossible See Chapter 8. See Chapter 8.
9.1.2 Pretreatments
Parameters
Permeabilization x Light microscopy Frozen-tissue sections Parafn-embedded sections y Electron microscopy Before embedding Without embedding After embedding 238
Facilitates the penetration of the reagents and the accessibility of the target nucleic acids Unnecessary Not indispensable, but sometimes useful Necessary, with thick sections See Chapter 8. See Chapter 8.
9.1 Deproteinization x Light microscopy Frozen-tissue sections Parafn-embedded sections y Electron microscopy Before embedding Without embedding After embedding Acetylation Storage x Light microscopy Frozen-tissue sections Parafn-embedded sections y Electron microscopy Before embedding Without embedding After embedding
Must be light to limit the risk of the diffusion of amplied products due to the destruction of cell structures Necessary; needs to be sufcient, without, however, affecting morphological preservation Necessary, with thick sections See Chapter 8. See Chapter 8. Often unnecessary Blockage of NH2 functions
Although difcult if pretreatments have been carried out, possible in anhydrous conditions Very difcult if pretreatments have been carried out; preferable to store blocks Impossible with this step See Chapter 8. See Chapter 8.

Parameters
Primer x Type Specic oligonucleotide Random oligonucleotides Poly (T) y Concentration +
Standard A risk of nonspecic hybridization of the primers on the large number of fragments of retrotranscribed cDNA The total cDNA retrotranscribed more accessible A minimal concentration of primers required to avoid the phenomenon of exhaustion The possibility of nonspecic hybridization Determination of the primer sequence so that its hybridization temperature is close to the optimal temperature for the activity of the transcriptase (between 40 and 42C, depending on the brand) 239
+++ z Hybridization temperature >42C 42C <42C
Controls and Problems Enzyme x Type y Concentration + +++ Temperature Duration <60 min 60 min >60 min
See Chapter 4. The risk of a reduction in the quantity of retrotranscribed cDNA due to exhaustion Between 40 and 42C, depending on the brand Threshold effect Standard Sometimes necessary in electron microscopy (with thick sections)
9.1.4 PCR
Parameters
Primers x Type Specic oligonucleotides y Concentration + +++ z Temperature Enzyme x Type y Concentration + +++ dNTP x Concentration + +++ y Type Direct PCR Conjugated dNTP Nonconjugated dNTP Indirect PCR Nonconjugated dNTP MgCl2 concentration Excess Insufciency 240
Standard A reduction in the efciency of the amplication The possibility of nonspecic hybridization Necessary that the two primers hybridize at temperatures that are very close together See Section 5.3.3.
A reduction in the efciency of the amplication The possibility of nonspecic DNA polymerase activity Depending on the manufacturer Standard: 200 M A possible reduction in amplication An increase in nonspecic reactions Standard Necessary to use the recommended respective proportions of conjugated and native dNTP Indispensable Indispensable Indispensable Must be optimized A reduction in the delity of the enzyme A reduction in the reaction yield
9.1 Temperature of the cycles x Denaturation <94C 94C >94C y Hybridization (TH) <TH TH >TH z Extension <72C 72C >72C Number of cycles <20 20 >20 Final extension <5 min 5 min >5 min
Necessary that the thermocycler be capable of producing rapid temperature changes in the sections 0 If the denaturation is partial, neither a PCR nor a RT-PCR can take place Standard Sometimes necessary (with thick sections) See Section 5.5.3. Threshold effect Standard Sometimes necessary to improve the specicity A reduction in the amplication reaction Standard Sometimes necessary Threshold effect Standard Sometimes necessary Not very effective Standard Often necessary Only in the case of indirect in situ PCR/ RT-PCR
9.1.5 Hybridization
Parameters
Probe x Type PCR product Oligonucleotides
Standard Chosen from the sequence of the amplied fragment according to the criteria set out in Chapter 6; the two probes must not interhybridize Background often a problem with radioactive hybridization A threshold effect in the detection process No saturation of targets Saturation of targets Increased possibility of nonspecic binding
y Label Radioactive Antigenic z Concentration + ++ +++
241
Controls and Problems Hybridization buffer x Salt concentration <600 mM 600 mM >600 mM y Dextran sulfate concentration <10% 10% >10% z tRNA, DNA concentration + ++ +++ { Detergent + ++ +++ Temperature Room temperature 37C >40C Duration <3 h 3h >5 h or
Reduces the stability of the hybrids Standard Increases the stability of the hybrids Reduces the concentration of the probes Standard Reduces the possibility of nonspecic bonds Reduces nonspecic bonds
Rarely useful Reduces the penetration of the reagents Not indispensable Favors the diffusion of the hybrids
Possibility of nonspecic hybridizations Reduction in nonspecic hybridizations Possibility of denaturation Limits background Often sufcient Possibility of improving the signal After PCR and/or hybridization
9.1.6 Washing
Parameters
NaCl concentration +++ + Temperature Room temperature >Room temperature Duration
Stability of the nonspecic hybrids Denaturation of nonspecic hybrids Washing nonspecic hybrids The denaturation of hybrids If the washing time too long, the hybrids can become unstable
9.1.7 Detection
Parameters
242
9.2 Autoradiographic x Macroautoradiography y Microautoradiography
Sensitivity/Specificity
Standard The possibility of quantitative estimation The type of developer, its optimal temperature, and the duration of the reaction will determine whether or not the signal stands out clearly from the background No penetration of the sections by the reagents Nonspecic adsorption An increase in sensitivity Little used; even after amplication, the signal not strong enough to permit direct observation Standard Essentially in electron microscopy
Immunocytological x Method Direct method Indirect method y Label Fluorescent
Enzymatic Particle
9.2
SENSITIVITY/SPECIFICITY
The optimization of a PCR/RT-PCR reaction consists of increasing its sensitivity, i.e., its lower threshold of detection, while ensuring its specicity.
9.2.1 Sampling
Parameters
Sensitivity 100 50 0 Specificity
Sampling conditions
The quality of the sampling, i.e., that of the preservation of the nucleic acids, has an inuence on the specicity of the reaction, since breaks can cause parasitic amplications. Ensures the preservation of nucleic acids, but also makes them much less accessible The preservation of morphology Indispensable step See Chapter 2. Standard Necessary that the xation conditions be known so that the different reactions can be adapted to them The shorter the xation process, the more accessible the nucleic acids 243
Fixation x Light microscopy y Electron microscopy z Type Formaldehyde Others { Duration +
Controls and Problems ++ +++ A compromise between cellular preservation and the diffusion of the amplied products In this case, more drastic pretreatments required A reduction in the diffusion of the amplied products Better accessibility of the nucleic acids A risk of increasing the diffusion of the amplied products Limits the diffusion of the amplied products Little used Standard The standard, for thick sections See Chapter 8. See Chapter 8. For the preembedding method If the storage conditions are appropriate, neither the sensitivity nor the specicity of the reaction will be adversely affected. 80C, or liquid nitrogen Necessary to store the slides in anhydrous conditions Virtually no time limit for conservation Not recommended; storage of blocks preferable Impossible See Chapter 8. See Chapter 8.
Freezing x Light microscopy Without xation
After xation y Electron microscopy Embedding x Light microscopy Parafn embedding y Electron microscopy Before embedding Without embedding After embedding Semithin sections Storage x Light microscopy Frozen samples Frozen sections Parafn-embedded samples Parafn-embedded sections y Electron microscopy Before embedding Without embedding After embedding
~ ~
9.2.2 Pretreatments
Parameters
Permeabilization x Light microscopy Frozen-tissue sections 244
Facilitates the penetration of the reagents and the accessibility of the target nucleic acids Unnecessary; and in any case this treatment too aggressive for frozen tissue sections
9.2 Parafn-embedded sections y Electron microscopy Before embedding Without embedding After embedding Deproteinization
Not indispensable; although it may sometimes be useful Necessary (with thick sections) See Chapter 8. See Chapter 8. Facilitates the accessibility of the nucleic acids enclosed in a network of proteins Necessary to be only slight if tissue destruction, which in the long run affects specicity, is to be limited Necessary; must be sufcient to ensure the unmasking of the nucleic acids without, however, affecting the preservation of the morphology Necessary (with thick sections) See Chapter 8. See Chapter 8. Often unnecessary Blockage of NH2 functions The storage of pretreated slides can only reduce the sensitivity and specicity of the reaction Even in anhydrous conditions, storage after pretreatment a delicate matter Storage of blocks preferable Impossible at this stage See Chapter 8. See Chapter 8.
x Light microscopy Frozen-tissue sections
Parafn-embedded sections y Electron microscopy Before embedding Without embedding After embedding Acetylation Storage
x Light microscopy Frozen-tissue sections Parafn-embedded sections y Electron microscopy Before embedding Without embedding After embedding

Parameters
Primer x Type Specic oligonucleotide
Specicity of the PCR determined by that of the RT
245
Controls and Problems Random oligonucleotides Numerous fragments of RNA are transcribed into cDNA, which means that the risk of nonspecic hybridization of the primers is increased. Can increase sensitivity in some cases Due to a reduction in the efciency of the RT The possibility of nonspecic hybridizations See Chapter 4. The quality of the enzyme very important for the two criteria in question Due to a reduction in the amount of nonspecic cDNA A possibility of nonspecic reverse transcription Due to a reduction in the efciency of the enzyme A possibility of nonspecic reverse transcription Differs according to the manufacturer Due to a reduction in the efciency of the enzyme By reducing the delity of the enzyme A reduction in the efciency of the enzyme Standard A longer incubation time has no effect. Sensitivity depends only on the quality of the enzyme.
Poly (T) y Concentration + +++ Enzyme x Type y Concentration + +++ z Cofactor MgCl2 + +++ Temperature + +++ Duration <60 min 60 min >60 min
9.2.4 PCR
Parameters
Primers x Type Specic oligonucleotides y Concentration + +++ 246
See Section 5.3.2; necessary to respect the criteria used to determine the specic primers A reduction in the efciency of the PCR The possibility of nonspecic hybridization
9.2 Enzyme x Type y Concentration + +++ dNTP x Concentration + +++ y Type Direct PCR Conjugated dNTP Nonconjugated dNTP Indirect PCR Nonconjugated dNTP Hot start Temperature of the cycles x Denaturation <94C 94C >94C y Hybridization (TH) <TH TH >TH z Extension <72C 72C >72C Number of cycles <20 20 >20 Final extension <5 min 5 min >5 min
See Section 5.3.3; the quality of the enzyme is crucial for both sensitivity and specicity Necessary to follow the manufacturers recommendations A possible reduction in amplication A possibility of nonspecic DNA polymerase activity Standard: 200 M A possible reduction in the amplication An increase in nonspecic reactions Standard The relative proportions of the two types of dNTP is crucial Indispensable Indispensable Indispensable See Section 5.5.2. The reliability of the thermocycler is important. The sample itself must be at the right temperature. A risk of false negatives Standard Sometimes necessary See Section 5.5.3. A threshold effect Standard Sometimes necessary to improve the specicity A reduction in the amplication reaction Standard Sometimes necessary A threshold effect Standard Sometimes necessary Not very effective Standard Often necessary
247
9.2.5 Hybridization
Parameters
Only in the case of indirect reactions
Probe x Type PCR product Single-stranded DNA Oligonucleotides y Label Radioactive Antigenic
Standard Limitation of the hybrids to one of the strands of the amplied product The two probes cannot interhybridize The sensitivity higher than with antigenic labels A threshold effect with the detection process A nonspecic signal, possibly due to endogenous enzymatic activities No saturation of the targets Saturation of the targets An increase in the possibility of nonspecic bonds Reduces the stability of the hybrids Standard Increases the stability of the hybrids Reduces the concentration of the probes Standard Reduces the possibility of nonspecic bonds Reduces the number of nonspecic bonds
z Concentration + ++ +++
Hybridization buffer x Salt concentration <600 mM 600 mM >600 mM y Dextran sulfate concentration <10% 10% >10% z tRNA, DNA concentrations + ++ +++ { Detergent + ++ +++ Temperature Room temperature
Rarely useful Favors the diffusion of the hybrids The possibility of nonspecic hybridizations
248
9.2 37C >40C Duration <3 h 3h >5 h or
A reduction in the number of nonspecic hybridizations Less chance of partial hybrids occurring Less background Often sufcient Signal better After direct PCR or hybridization
9.2.6 Washing
Parameters
NaCl concentration +++ + Temperature Room temperature >Room temperature Duration +++ +
Stabilization of specic and nonspecic hybrids Denaturation of nonspecic hybrids Elimination of nonspecic hybrids Partial denaturation of hybrids Must be optimized If the washing time is too long, possibility that the hybrids can become labile Limited elimination of partial hybrids
9.2.7 Detection
Parameters
Autoradiographic x Macroautoradiography y Microautoradiography Immunocytological x Method Direct method Indirect method y Label Fluorescent Enzymatic Particle
Standard 35 Cellular resolution with S
Standard Little used Standard Essentially in electron microscopy
249
9.2.8 Summary Table
Sample Criterion Sampling conditions Fixation x Type Paraformaldehyde Other y Duration + ++ +++ Freezing Light microscopy Without xation After xation Embedding x Parafn y Electron microscopy Before embedding Without embedding After embedding Semithin sections Storage x Light microscopy Frozen samples Frozen sections Parafn-embedded samples Parafn sections y Electron microscopy Before embedding Without embedding After embedding Signal Background Sensitivity Specicity
250
9.2 Pretreatments Criterion Deproteinization x Light microscopy Without embedding After parafn embedding y Electron microscopy Before embedding Without embedding After embedding Permeabilization x Light microscopy Without embedding After parafn embedding y Electron microscopy Before embedding Without embedding After embedding Acetylation Prehybridization x Light microscopy Without embedding After parafn embedding y Electron microscopy Before embedding Without embedding After embedding Storage x Light microscopy Without embedding After parafn embedding y Electron microscopy Before embedding Without embedding After embedding Signal Background
Sensitivity
Specicity
251
Controls and Problems Reverse transcription Criterion Primer x Type Specic oligonucleotide Nonspecic oligonucleotide Poly (T) y Concentration + +++ Enzyme x Type y Concentration + +++ z Cofactor + +++ Temperature + +++ Duration <60 min 60 min >60 min PCR Criterion Primer x Type Specic oligonucleotide y Concentration + +++ Signal Background Sensitivity Specicity Signal Background Sensitivity Specicity
252
9.2 Enzyme x Type y Concentration + +++ dNTP x Concentration + +++ y Type Direct PCR Conjugated dNTP Nonconjugated dNTP Indirect PCR Nonconjugated dNTP Hot start Temperature of the cycles x Denaturation <94C 94C >94C y Hybridization (TH) <TH TH >TH z Extension <72C 72C >72C Number of cycles <20 20 >20 Final extension <5 min 5 min >5 min 0
253
Controls and Problems Hybridization Criterion Probe x Type PCR product Single-stranded DNA Oligonucleotide y Label Radioactive Antigenic z Concentration + ++ +++ Hybridization buffer x Salt concentration <600 mM 600 mM >600 mM y Dextran sulfate concentration <10% 10% >10% z tRNA, DNA concentration + ++ +++ { Detergent + ++ +++ Temperature Room temperature 37C >40C Signal Background Sensitivity Specicity
254
9.2 Duration <3 h 3h >5 h or
or
Washing Criterion NaCl concentration +++ + Temperature Room temperature >Room temperature Duration +++ + Signal Background Sensitivity Specicity
Detection Criterion Autoradiographic Macroautoradiography Microautoradiography Immunocytological x Method Direct method Indirect method y Label Fluorescent Enzymatic Particle Signal Background Sensitivity Specicity
255
9.3
CONTROLS
9.3.1 Tools and Reagents

Parameters
Positive Negative
Primers Search for homologous sequences in a gene bank. Probes For the hybridization procedure Specic reagents for amplication Carrying out a liquid-phase PCR for a commonly expressed gene (e.g., actin), using the same reagents Reverse transcription Carrying out a liquid-phase RT for an RNA expressed in all the types of tissue (e.g., actin), using the same reagents Immunocytological reaction Using the same reagents to detect a protein that is present in the tissue being studied
Signicant homologies between sequences can result in the formation of various amplied products, or the amplication of a product that is common to several genes. Indirect PCR/RT-PCR The existence of signicant homologies between sequences can result in the formation of hybrids that are unwanted but specic to the probes. The presence of a band on the electrophoresis gel means that all the reagents (i.e., the enzyme, buffer, MgCl2, and nucleotides) are usable for the in situ reaction. The presence of a band on the electrophoresis gel means that all the reagents (i.e., the enzyme, buffer, cofactor, and nucleotides) are usable for the in situ reaction. A positive reaction means that all the reagents (i.e., the secondary antibody and the chromogen) are usable for the in situ reaction. All the reagents (i.e., the primary and/or secondary antibodies, and the chromogen) can be tested to detect a standard in situ hybridization. If necessary, check that there is no endogenous enzymatic activity.
256
9.3
Controls
9.3.2 Tissue
Parameters
Positive Negative
Positive tissue Negative tissue Internal control
Heterogenous culture Coculture Heterogeneous tissue Sections of two different types of tissue Destruction of the target by treatment DNase RNase
Indispensable Tissue or cells known to express the target nucleic acid Indispensable Tissue or cells known not to express the target nucleic acid One of the best controls Positive and negative cells in the same tissue or culture Several cell types (e.g., a primary culture) For example, two cell lines The most common case For the same reaction
an enzymatic The breakdown of DNA before the in situ PCR Necessary to follow the breakdown of RNA by abundant washes before the reverse transcription step The breakdown of DNA before the in situ RT-PCR, to make sure that there is no amplication of the genomic DNA The breakdown of RNA before the in situ PCR
Destruction of nontarget nucleic acids by enzymatic treatment DNase
RNAse
9.3.3 Pretreatments
Parameters
Positive Negative
Fixation
An in situ hybridization reaction using poly (T) probes shows that mRNA remains in situ.
257
Controls and Problems Deproteinization The compatibility of this deproteinization step with the preservation of morphology and of the nucleic acids can be checked by a classical in situ hybridization using a poly (T) probe or a probe corresponding to highly expressed RNA or DNA. The compatibility of this permeabilization step with the preservation of morphology and that of the nucleic acids can be checked by classical in situ hybridization. The destruction of these nucleic acids (RNA or DNA) can be checked by classical in situ hybridization.
Permeabilization
The destruction of target nucleic acids. RNAse or DNAse pretreatment

Parameters
Positive Negative
Omission of the enzyme
Omission of the primer Omission of the dNTP Pretreatment with RNase Pretreatment with DNase
This should abolish the signal. A slightly positive reaction may, however, be observed, corresponding to the signal that would be obtained with hybridization alone. A simple negative control that gives the same result as before. This is an unsatisfactory control. The dNTP in the cells is sometimes sufcient to give a positive reaction. A positive result with the in situ RT-PCR indicates a nonspecic reaction. No modication of the reaction should be observed if the DNase is RNase-free. A positive result can be seen as the amplication of genomic DNA. The washes following the pretreatment must be carried out with great care.
258
9.3
Controls
9.3.5 PCR
Parameters
Positive Negative
Omission of the enzyme
Omission of the primers
Omission of the dNTP
Omission of the labeled dATP Pretreatment with RNAse In situ PCR reaction In situ RT-PCR reaction Pretreatment with DNAse In situ PCR reaction In situ RT-PCR reaction
This should abolish the signal, although a slightly positive reaction may be observed, corresponding to the only hybridization of the retrotranscribed RNA and/or the preexisting DNA. A positive reaction with the in situ PCR/ RT-PCR corresponds, in this case, to the DNA polymerase activity of the enzyme, obtained from breaks in the DNA. In this case the fault may lie with the xation step. A positive reaction does not invalidate the in situ PCR/RT-PCR amplication. The dNTP in the tissue may be sufcient to produce amplication. This is done only in the case of direct in situ PCR/RT-PCR. No modication of an in situ PCR should be detectable if the RNAse is DNAse-free. This should abolish the signal. A positive in situ RT-PCR indicates a nonspecic reaction. This should abolish the signal. A positive in situ PCR indicates a nonspecic reaction. No modication of the RT-PCR should be observed if the DNAse is RNAse-free. The washes following the pretreatment must be carried out with great care. Only with indirect in situ PCR/RT-PCR
9.3.6 Hybridization/Washing
Parameters
Positive Negative
Labeling the probe Radioactive Antigenic
Determination of the specic activity Dot on membrane
259
Controls and Problems Omission of the probe Hybridization with a heterologous probe of the same type Action on the efciency of the hybridization reaction Action on the stringency of the washes If the reaction is positive, the presence of an endogenous label (e.g., biotin, or an enzyme) must be suspected. The specicity of the detection of the amplied product A variation in the hybridization temperature induces a modication of the signal. A variation in the salt concentration or the temperature of the washes induces a modication of the signal.
9.3.7 Detection
Parameters
Positive Negative
Autoradiographic Immunohistological Omission of the primary antibody Omission of the conjugated antibody
Determination of the background A negative reaction A search for endogenous activity: Endogenous biotin Endogenous peroxidase Endogenous alkaline phosphatase All positive reactions can be inhibited by specic chemical compounds (e.g., hydrogen peroxide or levamisol). A positive reaction reveals the presence of uorescence or an induced stain.
Omission of the uorescent label or chromogen
9.3.8 Results
Parameters
Positive Negative
Signal/background ratio
Indispensable (see Section 9.3.2)
260
9.3 Reproducibility Extraction of the amplied product from a section Reaction medium
Controls
A similar location of the signal in two adjacent sections, or a section from another sample processed in the same conditions. With migration on a gel, a band should appear at a predetermined position on the column. Sample the reaction medium for the amplication step, and if, after electrophoresis, a band corresponding to the size of the amplied product appears, this means that a large amount of diffusion has taken place, in which case the possibility of a false positive must be considered, and the experimental conditions revised (see Section 9.4).
9.3.9 Validation by Other Techniques

Parameters
Positive Negative
Liquid-phase PCR Liquid-phase RT-PCR Immunocytology Transgenic animals Knockout animals
Demonstration of the presence or absence of the target DNA in the tissue under consideration Demonstration of the presence or absence of the target RNA in the tissue under consideration Detection of the protein transcribed from the target nucleic acid Overexpression of the gene being sought Suppression of the gene being sought
261
9.3.10
Summary Table
Methods Controls Positive Negative Internal control Adjacent sections Destruction of the targets Pretreatments Direct + 0 + + 0 PCR Indirect + 0 + + 0 + 0 + + 0 RT-PCR Direct Indirect + 0 + + 0
Modication
Tissue
Fixation Deproteinization Permeabilization Reverse transcription Liquid-phase reaction
+/ +/ +/
+/ +/ +/
+/ +/ +/ + 0 0 +/
+/ +/ +/ + 0 0 +/ + 0 0 0 +/ + 0 0 +/ +
Omission
Enzyme Primer dNTP Amplication Liquid-phase reaction Taq DNA polymerase + 0 0 0 0 +/ + 0 0 +/ + +/ + 0 0 +/ + + 0 0 0
+ 0 0 0 0 +/ + 0 0 +/ +
Omission
Primers Nonconjugated dNTP Conjugated dNTP Stringency of washes Hybridization Positive tissue Omission of probes Heterologous probe Stringency of washes Detection Positive control
262
9.4 Omission Primary antibody Secondary antibody Chromogen Results Reproducibility Signal/background ratio Reaction medium Other techniques +
Positive result
False Positives 0 0 0 + 0
0 0 0 + 0
0 0 0 + 0
0 0 0 + 0
+/
Variable result
Negative result
9.4
FALSE POSITIVES
This is a signicant risk in in situ PCR/ RT-PCR. See Section 9.3.9. It is not always a problem of specicity strictly speaking, but of a parallel reaction brought about in the course of the in situ amplication. This is possible when all the different types of tissue are positive, including the tissue used as a negative control. This is possible only with direct reactions. From xation to pretreatments, there are numerous possible causes for breaks in DNA. Taq DNA polymerase becomes active at 40C, attaching to a 3 hydoxylated end. This mostly concerns the primers that dene the PCR. This risk should not be overlooked (see Chapter 5). Among the different causes of false positive, this is the most difcult to detect. This is normally a low risk.
False positives are due to suspect controls, which do not allow the signal to be identied as specic to the amplication of the sequence of interest. The causes are relatively few in number:
The nonspecic synthesis of the sequence of interest can result from: The nonspecic incorporation of the label The repair of cellular DNA Nonspecic amplication Nonspecic hybridization of the primers Amplication of genomic DNA Diffusion of the amplied products External contamination It is also necessary to check for false positives that might result from less fundamental causes: Problems due to the specicity of the reagents Nonspecic detections
Handling errors Artifacts, etc.
These can be due to endogenous enzymatic activity and to nonspecic tools (antibodies, chromogens). Recommence the reaction. Recommence the reaction. 263
9.4.1 Nonspecic Incorporation of Labels

The labels are carried either by: Labeled dNTP, or Labeled primers, used in direct in situ PCR or RT-PCR 9.4.1.1 Denition of the problem See Section 9.3. An absence of negative cells See Section 9.3. Essentially dUTP or dATP (see Section 5.3.1) See Section 5.3.2.7.
This may be the problem when a signal: Appears in tissue dened as negative Is found in all the cells in the section being studied Is not inhibited by a control carried out in the absence of primers 9.4.1.2 Causes A consequence of the nonspecic hybridization of primers A consequence of insufcient washing
There are two main causes: Nonspecic synthesis The presence of residual dNTP or labeled primers 9.4.1.3 Solutions
There are several possible solutions, depending on the strength of the signal. Use the indirect method. Increase the stringency and duration of the washes. Increase the hybridization temperature of the primers. The disappearance of the labeled primers or dNTP eliminates this problem. This eliminates the labeled primers or dNTP not incorporated into the amplied products. This potentially reduces nonspecic amplications.
9.4.2 Repair of Cellular DNA

Nucleic acids undergo modications, destruction, and repair. And, in fact, breaks are sometimes used as markers (e.g., apoptosis). The repair of breaks in the presence of dNTP and an enzyme is thus a phenomenon that must not be overlooked. Breaks can be revealed by specic labeling (i.e., in situ 3 extension). The risk concerns only direct PCR/ RT-PCR reactions. The label must be carried by dNTP. The problem does not exist if the label is carried by the primer.
264
9.4 9.4.2.1 Denition of the problem
False Positives
There is an amplied signal, but also nonspecic labeling. In the absence of an enzyme, these two signals disappear. In the absence of primers, on the other hand, this nonspecic labeling persists, whereas the amplied signal disappears. 9.4.2.2 Causes
DNA polymerase acts both in the amplication reaction and in the repair of breaks. The signals do not appear in the absence of enzyme. No amplication takes place in this case, but the repair of breaks remains possible.
Taq DNA polymerase acts on breaks in DNA. 9.4.2.3 Solutions
This property can be used for in situ labeling.
The destruction of the genomic DNA before amplication An indirect reaction
For pretreatment with DNase, see Section 3.7.3. This repair can also take place during an indirect reaction, but is not detected during the hybridization step.
9.4.3 Nonspecic Synthesis

Any synthesis not corresponding to the amplied product is considered to be nonspecic. 9.4.3.1 Denition of the problem Detected by suspect controls Note: This signal may be superimposed on the specic signal. The polymerase activity disappears. This test is the most convincing. This synthesis can appear in a random way in any of the cells. This nonspecic signal can be superimposed on a specic signal.
This signal disappears if the enzyme is omitted. Negative tissue appears positive. There is no internal control. The signal is not reproducible on two adjacent sections. 9.4.3.2 Causes
This type of synthesis can have different causes: Amplication common to several genes
Nonspecic hybridization of the primers. Repair of breaks with: Incorporation of labeled nucleotides Hybridization of certain random sequence regions with probes intended for the detection of the amplied products.
They are given in decreasing order of importance. Verication of the absence of homology between the primer sequences and one or more sequences within the genome Particularly high risk The synthesis of random sequences Labeling during synthesis (direct method) Minimal risk
265
Controls and Problems 9.4.3.3 Solutions An increase in the specicity of the PCR step Only the amplied products detected by the hybridization step An increase in the specicity of the detection step
An increase in the hybridization temperature of the primers during the PCR cycles An indirect reaction An increase in the hybridization temperature of the probes
9.4.4 Nonspecic Hybridization of the Primers

This is a crucial phase in the reverse transcription step and the PCR cycle, as the hybridization of the primers determines the specicity of the reaction. 9.4.4.1 Denition of the problem One of the best controls for detecting false positives In the case of an internal control One solution to the problem Hybridization errors result in nonspecic syntheses.
Tissue that is normally negative is in fact positive. In heterogeneous tissue, all the cells are positive. Stringent washes abolish this signal. 9.4.4.2 Causes
Nonspecic hybridization of the primer specic during the RT step can result in the synthesis of DNA of different sizes. Nonspecic hybridization of the primers for the PCR step leads to the synthesis of strands of DNA that do not correspond to the nucleic acid sequence of interest. 9.4.4.3 Solutions
The efciency of the PCR will then be lower, but this lack of specicity cannot, on its own, explain a false positive. Their nucleotide sequences and Tm may be responsible for this.
Change the primers.
Increase the hybridization temperature(s) of the PCR cycle. Increase the stringency of the washes to denature partial hybrids.
Do this where only the amplication is nonspecic. Check the potential complementarity of their sequences against a gene bank. Check that the two primers hybridize at very similar temperatures. See Section 5.5.7.
9.4.5 Amplication of Genomic DNA

This is the most common type of false positive. The signal is generally limited to the nuclear compartment, and can be present in any of the cells. All the cells in an organism have the same genome. This risk is high, and the experimenter must be very careful about the choice of the primers and their position on the gene in relation to the introns.
266
False Positives
All the nuclei are positive.
No reaction-extinction control is conclusive. The controls for the reverse transcription step remain positive. The controls for the amplication step are correct. 9.4.5.2 Causes
It must also be remembered that the nuclear signal can have different origins (e.g., a nuclear virus, a detection problem, or the diffusion of amplied products into the nuclear compartment). See Section 9.3. See Section 9.3. The reverse transcription can be effective, and can lead to specic amplication. See Section 9.3. This type of amplication is specic.
There are at least two copies of the gene of interest in the genomic DNA. The primers are incorrectly positioned on the structure of the gene. 9.4.5.3 Solutions
Some authors claim that amplication can take place with just one copy.
A change of primers, so that this type of genomic amplication becomes impossible Verication of the new primers Destruction of the genomic DNA before amplication
See Section 5.3.3.2. By liquid-phase PCR Pretreatment with DNase (see Section 3.7.3)
9.4.6 Diffusion of Amplied Products

The amplied products are obtained from sequences of nucleic acids in cell structures consolidated by xation. Given the permeabilization necessary to the penetration of tools and reagents, it is important to check that an amplied product has not diffused, rst into adjacent cells, and second into the reaction medium, where it can be eliminated by washing. It is sometimes said that the multiplication of the amplied products could cause them to move away from the original target sequence. 9.4.6.1 Denition of the problem There can be so much diffusion that all the tissue appears positive. See Section 9.3. This is the best control for evaluating this particular risk. In theory, the risk of false positives resulting from the diffusion of an amplied product is considerable, but in practice it seems minor.
This remains hypothetical.
The signal diffuses along a decreasing gradient from the most to the least positive cells. The amplication controls are positive. The internal control is negative.
267
Controls and Problems 9.4.6.2 Causes Poor xation, or overaggressive pretreatments The most common cause
Partial or total destruction of cell or tissue structures Faulty xation 9.4.6.3 Solutions
An improvement in the xation conditions An increase in the stringency of the washes
A reduction in the pretreatments, and in particular the permeabilization and deproteinization steps.
To reinforce the cell and tissue structure The amplied products that have diffused are seldom if ever attached to cell or tissue structures, and are easy to eliminate. Making sure that the PCR reaction is not, however, inhibited; a satisfactory compromise may be difcult to nd This is a minor theoretical risk, as the tissue is an entity in itself. It can occur at any level.
9.4.7 External Contamination

External contamination can produce a positive result in tissue. However, it depends on such an unlikely combination of preconditions that for practical purposes it does not constitute a risk of false positives. 9.4.7.1 Denition of the problem
A homogeneous signal is observed over the whole section, or along a gradient which decreases from the periphery inward. All the controls of specicity are positive, but the negative control is positive. The internal control allows this risk to be evaluated. The result is not reproducible from one slide to another. 9.4.7.2 Causes
Contamination by the external medium. See Section 9.3. The signal is specic. Only internal controls (i.e., negative cells) can provide hard evidence of contamination.
The presence of target sequences external to the sample, which contaminate: Equipment Sections Solutions 9.4.7.3 Solutions
Sequences deriving from previous reactions While they are being cut While they are being prepared If the cause is not identied, all possible precautions must be taken. Pipettes, single-use articles, etc.
Replacement of all primers, enzymes, reagents, etc. Replacement of all the minor equipment
268
9.5 Replacement of all the preparations Decontamination of the work surfaces Decontamination of the thermocycler
False Negatives
Fixatives, and in particular the buffers The laboratory Separation of the working areas With some models, this is easy
9.4.8 Summary Table

Nonspecic synthesis
Cellular DNA repair
Amplication of the genomic DNA
Nonspecic incorporation of the label
Nonspecic hybridization of the primers
Diffusion of the amplied products + + + +
Causes
Controls Positive Tissue Negative Internal control Adjacent sections Destruction of the targets Pretreatments Taq DNA polymerase Omission Reverse transcription Primers Hybridization Detection Stringency of the washing +
+ +
+ + + +
+ +
+ + + +
+ +
+ + + + +
+ +
+ +
False positive Controls to determine the cause of a false positive.
9.5
FALSE NEGATIVES
All the controls carried out in the liquid phase must be positive. The aim of this technique is to identify nucleic acid sequences that are very weakly expressed, and can thus very easily be lost during: The preparation of the sample The stabilization of the structures The pretreatments 269
These are characterized by the result of a negative in situ PCR/RT-PCR on tissue known to be positive. False negatives are much less frequent than false positives. Among their causes are the following: The destruction of target sequences Fixation problems Digestion problems
External contamination + + + + + + +
Controls and Problems Flaws in the reverse transcription process These are due to a problem with the hybridization of the primer, or a malfunctioning enzyme. These are due to a problem with the hybridization of the primer, or a malfunctioning enzyme. Elimination of the amplied products With more than 30 cycles, it is improbable that a specic result will be obtained. See Section 9.3.
Flaws in the amplication process
Overabundant washes The rst option is to increase the number of cycles. One must, however, exclude false negatives caused, for example, by: The quality of the reagents Handling errors Artifacts
Recommence the reaction. Recommence the reaction.
9.5.1 Destruction of Target Sequences

These sequences, of which there are only a few copies in a handful of cells, can be destroyed, in particular, during sampling or pretreatment if certain conditions are not satised. 9.5.1.1 Denition of the problem The only evidence of false negatives in situ This is a major theoretical risk, in particular for RNA sequences (i.e., for RT-PCR methods). If the sampling is not rapid enough, intracellular autolysis may occur.
The absence of a signal in the positive control tissue. The amplication reaction carried out on the same sample after the extraction of the nucleic acids is: Either equally negative, or Positive
The sample must be responsible. The in situ manipulation must be responsible.
9.5.1.2
Causes Intracellular autolysis Contamination by RNase or DNase from the equipment or the operator A problem, in particular, with frozen samples
Overlengthy sampling times Nonsterile sampling conditions Storage of samples
9.5.1.3
Solutions If the sample comes from a retrospective series, look at the way the sample was processed. It is possible that the sample was not processed in conditions favorable to the conservation of nucleic acids.
The sampling conditions and protocol will need to be reviewed. Check for the presence of RNA before carrying out an in situ RT-PCR reaction by in situ hybridization with a poly (T) probe.
270
9.5
False Negatives
9.5.2 Problems Related to the Fixation Process

This step is indispensable to the morphological preservation and stabilization of structures, but it often results in the partial destruction of the target sequences. Prolonged xation can destroy or mask target sequences, while, if it is reduced to the minimum, it results in false positives (see Section 9.4). A compromise must be found for each type of tissue studied, on a case-by-case basis.
9.5.2.1
Denition of the problem The only evidence of false negatives in situ Positive PCR/RT-PCR in liquid phase Necessary to consider the trade-off between morphological preservation and the preservation of target nucleic acids (see Chapters 2 and 3). See Chapter 2; some xatives incompatible If this is negative, there is no more mRNA in the tissue, and it is likely that the sequence of interest has also disappeared (see Section 2.1.3.3). These are often simple, and result from a lack of information about how the sample was prepared. This is by diffusion, if the xation is insufcient. The target sequences are masked, and are difcult to access. Some xatives cause breaks in the target sequences, others modications of the bases (see Chapter 2). These are generally very simple. Indispensable If possible, apply the standard conditions (see Chapter 2). This reduces the cross-linkages created by the xative between the nucleic acids and the proteins (see Chapter 3).
Signal is absent in the positive control tissue. A positive amplication reaction is obtained on the same sample after the extraction of the nucleic acid. The morphology of the tissue is excellent.
The xation conditions and/or the type of xative are not known. Carry out a hybridization with a Poly (T) probe.
9.5.2.2
Causes
Target sequences are lost. The connement of target sequences in a molecular framework is more or less marked according to the duration of the xation process. The target sequences are modied by the xative.
9.5.2.3
Solutions
Check the xation conditions. Change the xation conditions. Change the pretreatments.
271
9.5.3 Problems Related to Proteasic Digestion

This step is indispensable to making the nucleic acid accessible and facilitating the penetration of the tools and reagents. 9.5.3.1 Denition of the problem The only evidence of false negatives in situ Positive PCR/RT-PCR in liquid phase Often results in poor preservation of the target nucleic acid This test demonstrates the diffusion of the amplied product. The diffusion is minimal, and does not explain the false negative. Risks due to pretreatments (see Chapter 3)
An absence of signal in the positive control tissue A positive result for an amplication reaction carried out on a sample after the extraction of the nucleic acid Poor preservation of tissue morphology The reaction mixture analyzed after in situ amplication: A band of the expected size appears on the electrophoresis gel A band of the expected size appears after a PCR or a nested PCR 9.5.3.2 Causes
The loss of target sequences
The loss of cDNA The loss of amplied products
By diffusion from tissue made overpermeable by pretreatments (e.g., deproteinization or permeabilization) By diffusion during washing after reverse transcription By diffusion during washing after amplication
9.5.3.3
Solutions Conservation of the target sequences in the cell May be necessary to change the brand or the batch Step needs to be optimized (see Section 3.5.2.1) Conservation of the cell structures A reduction in the diffusion of the amplied products
Cut down the deproteinization step by reducing: The proteinase K concentration The incubation time Change the xation conditions. Reduce, or cut out altogether, the permeabilization step.
9.5.4 Reverse Transcription Problems

This is a necessary step that precedes the amplication step. Without it, no amplication of RNA would be possible. Limited to in situ RT-PCR methods This is the most difcult step to control, and is probably the one that gives rise to the largest number of unexplained negative reactions.
272
False Negatives
The possibility of reverse transcription problems is generally considered only after other potential problems have been ruled out. An absence of signal in the positive control tissue A control carried out in the liquid phase after the extraction of the nucleic acid from the same sample: Positive reaction
This is the only evidence of false negatives in situ.
Negative reaction
The RT conditions are good (enzyme, concentration); the problem has to do with the in situ adaptation. This is a genuine false negative. The problem with the RT will be difcult to identify. There are a number of potential causes. Essentially a problem of concentration Essentially a problem of concentration, although its effectiveness, age, and storage conditions should also be checked Necessary to optimize its concentration Unlikely to be responsible if the reaction is positive in the liquid phase That is, the temperature, the duration, and the programming of the thermocycler
9.5.4.2
Causes
The primer The enzyme
The cofactor of the enzyme The reagents The experimental conditions
9.5.4.3
Solutions That is, the enzyme, the primer, and the cofactor Lower the hybridization temperature, even though this involves the risk of a nonspecic signal being generated. The usual risks, especially for an inexperienced operator, as all the steps must be optimized The equipment, programming, handling, reaction medium, reagents, primers
Increase the concentrations of the different reagents. Modify the thermocycler settings.
9.5.5 Amplication Problems

There are a number of potential sources of problems. 9.5.5.1 Denition of the problem
An absence of signal in the positive control tissue A control carried out on the same sample after the extraction of the nucleic acid: Positive reaction
This is the only evidence of false negatives in situ. The in situ adaptation alone must be responsible. This is a genuine false negative. 273
Controls and Problems Negative reaction It is not possible to determine the origin of the problem. It will be necessary to carry out a PCR on a strongly expressed target sequence to test the experimental conditions, the reagents, the thermocycler, etc. The lack of experience of the experimenter can be ruled out. Numerous, and often cumulative The temperatures of the sections do not correspond to the indicated temperatures. The programming of the temperatures and durations of the different phases of the cycle is faulty. There are bubbles in the reaction medium. The hybridization phase of the cycle does not take place properly. The enzyme is adsorbed, which means that no amplication occurs. The cofactor is adsorbed, which means that no amplication occurs. No amplication can take place.
The reaction remains negative after several assays, whereas other reactions were positive. 9.5.5.2 Causes
A malfunctioning of the thermocycler
A handling error Incompatible hybridization temperatures of the two primers Insufcient amount of enzyme The MgCl2 concentration too low PCR chamber adsorbing the reagents 9.5.5.3 Solutions
It is difcult to calibrate the thermocycler. It is, however, possible to test the temperature of the three fundamental stages of the PCR cycle. Denaturation temperature
Test another thermocycler if possible. Test with a known positive reaction in liquid phase. Amplication of strongly expressed test DNA. A genuinely negative result may indicate that the denaturation temperature has not been attained. Hybridization using an oligonucleotide probe with a low Tm; a negative result indicates that the temperature was too high. Whatever the temperature difference, the amplication reaction is weak, but not negative. These reactions are very sensitive, and a lack of attention often produces a negative (or false-positive) result. See Chapter 4. It is possible to extend the primer with the lowest Tm so as to increase it. See Chapter 5. Commercial products are ready to use. If cover slides are used, it is recommended that they be siliconized and sterilized (see Appendix A3.2).
Hybridization temperature
Extension temperature Repeat the reaction to eliminate risks resulting from the experimenter. Check the Tm of the primers.
Make a range of concentrations of the enzyme and of MgCl2. Pretreat the components of the reaction chamber.
274
9.5
False Negatives
9.5.6 The Stringency of the Washes

The stringency of the washes must be adapted to the experiments on a case-by-case basis. It should be increased only to reduce nonspecic signals.
The risk is all the higher, as the amplication is weak. To adjust the stringency, it is preferable to choose conditions that give a signal, even if there is a signicant amount of background, which can be eliminated in a number of ways. To start out from a negative result is always more difcult.
9.5.6.1
Denition of the Problem The only evidence of false negatives in situ. Positive PCR/RT-PCR in liquid phase. Poor preservation of the structures favors the elimination of the amplied products. The amplication has in fact taken place.
An absence of signal in the positive-control tissue. A positive amplication reaction carried out on the same sample after the extraction of the nucleic acid. The morphological preservation is not satisfactory. A nested PCR carried out on an aliquot of the reaction mixture sampled after the amplication should be positive. 9.5.6.2 Causes
Numerous, and often cumulative. Especially if the amplication is weak.
Elimination of the amplied products. 9.5.6.3 Solution
Reduce the stringency of the washes.
Limit the washes to one or two baths of 2X SSC. Rare. It must be checked on a positive model.
9.5.7 Detection Problems

Detection is now a well-worked-out step, both in immunocytology and autoradiography. 9.5.7.1 Denition of the problem
An absence of signal in the positive-control tissue. A positive amplication reaction carried out on a sample after the extraction of the nucleic acids. There is no background. 9.5.7.2 Causes
The only evidence of false negatives in situ. PCR/RT-PCR in positive liquid phase. There may be exceptions. Occasional. The most common. The detection step was not carried out.
Handling errors. Antibodies, chromogens, or nuclear emulsions beyond their expiration date.
275
Controls and Problems 9.5.7.3 Solutions See Chapter 7. Using another technique (e.g., immunocytology) See Chapter 7.
Review the protocol. Check the reagents. Recommence the detection process with another protocol.
9.5.8 Summary Table

Destruction of the target sequences Amplication problems Pretreatments Stringency of the washes Reversetranscription problems Causes Controls Positive Negative Internal control Adjacent sections Destruction of the targets Pretreatments Taq DNA polymerase Omission Reverse transcription Primers Hybridization Detection Stringency of the washes
Negative control
Tissue
Fixation
Controls to determine the cause of a false positive.
276
Chapter 10
Typical Protocols
Contents
CONTENTS
10.1 Direct in Situ PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1.1 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 10.1.2 Typical Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1.2.1 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1.2.2 Preparation of Samples . . . . . . . . . . . . . . . . . . . . . . 10.1.2.2.1 Cell Cultures . . . . . . . . . . . . . . . . . . . . 10.1.2.2.2 Frozen Fixed Tissue . . . . . . . . . . . . . . . 10.1.2.2.3 Fixed Parafn-Embedded Tissue. . . . . . . . . . . . . . . . . . . . . . . . . . 10.1.2.3 Pretreatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1.2.3.1 Cell Cultures and Frozen Sections . . . . . . . . . . . . . . . . . . . . . . . . 10.1.2.3.2 Parafn-Embedded Fixed Tissue. . . . . . . . . . . . . . . . . . . . . . . . . . 10.1.2.4 In Situ Amplication . . . . . . . . . . . . . . . . . . . . . . . . 10.1.2.5 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1.2.6 Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 Indirect in Situ PCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.1 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 10.2.2 Typical Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.2.1 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.2.2 Preparation of Samples . . . . . . . . . . . . . . . . . . . . . . 10.2.2.3 Pretreatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.2.4 In Situ Amplication . . . . . . . . . . . . . . . . . . . . . . . . 10.2.2.5 Hybridization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.2.5.1 Labeling Probes by Extension of the 3 End . . . . . . . . . . . . . . . . . . . . 10.2.2.5.2 Hybridization . . . . . . . . . . . . . . . . . . . . 10.2.2.6 Revelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.2.7 Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3 Indirect in Situ RT-PCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3.1 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 10.3.2 Typical Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3.2.1 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3.2.2 Preparation of Samples . . . . . . . . . . . . . . . . . . . . . . 10.3.2.3 Pretreatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3.2.4 DNase Treatment. . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3.2.5 Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . 10.3.2.6 In Situ Amplication . . . . . . . . . . . . . . . . . . . . . . . . 10.3.2.7 Hybridization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3.2.8 Revelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3.2.9 Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.4 Direct in Situ RT-PCRSpecial Case: Cell Suspension. . . . . . . . . . . . 10.4.1 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 10.4.2 Typical Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.4.2.1 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281 281 282 282 282 282 283 283 284 284 285 285 287 288 289 289 290 290 290 290 291 291 291 292 293 294 294 295 295 295 296 296 296 297 297 298 299 300 300 301 302 302 279
Typical Protocols Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pretreatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . Amplication with Labeled Primers . . . . . . . . . . . . 10.4.2.5.1 Fluorescent Primers . . . . . . . . . . . . . . . 10.4.2.5.2 Biotinylated Primers . . . . . . . . . . . . . . 10.4.2.6 Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5 Indirect in Situ RT-PCR on Vegetable Tissue Using Floating Vibratome Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5.1 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 10.5.2 Typical Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5.2.1 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5.2.2 Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5.2.3 Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5.2.4 Pretreatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5.2.5 Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . 10.5.2.6 Amplication. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5.2.7 Hybridization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5.2.8 Immunocytological Detection . . . . . . . . . . . . . . . . . 10.5.2.9 Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5.2.10 Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6 Indirect in Situ RT-PCR Using Electron Microscopy . . . . . . . . . . . . . . 10.6.1 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 10.6.2 Typical Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6.2.1 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6.2.2 Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6.2.3 Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6.2.4 Pretreatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6.2.5 Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . 10.6.2.6 Amplication. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6.2.7 Hybridization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6.2.8 Embedding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6.2.9 Ultramicrotomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6.2.10 Immunocytological Detection . . . . . . . . . . . . . . . . . 10.6.2.11 Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6.2.12 Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.4.2.2 10.4.2.3 10.4.2.4 10.4.2.5 303 304 304 305 305 306 308 308 309 310 310 310 311 311 311 312 313 314 314 315 315 316 316 316 317 317 318 318 318 319 320 321 321 321 321
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10.1 When using in situ PCR and RT-PCR methods, the idea of typical protocols represents something of a challenge. The protocols given in this chapter should help beginners save time and avoid the main pitfalls. These recipes do, however, need to be adapted and adjusted to the researchers personal objectives, and in the end it is only through a process of trial and error that satisfactory results will be obtained. A positive result cannot be taken for granted unless controls are carried out.
Direct in Situ PCR
See Chapter 9.
10.1 DIRECT IN SITU PCR

Direct in situ PCR is essentially used to detect viral DNA or identify particular genes in cultured cells, frozen xed tissue, or parafn-embedded tissue sections.
See Chapter 11, Figures 11.1 and 11.2.
Gloves must be worn. All the products must be RNase-free, the solutions must be prepared in DEPC water (see Appendix B1.2), and all equipment must be sterilized (see Appendix A2).
281
Typical Protocols
10.1.2 Typical Protocols

10.1.2.1 Solutions See Appendix B6.2.2.1. See Appendix B6.2.1. dNTP
BCIP 0.4 mM biotin-14-dATP Blocking agents 10 mM dTTP, dCTP, dGTP, dATP Ethanol 100 (20C) Ethanol 70, 95, 100 Isopentane Liquid nitrogen 50 mM MgCl2 9 NaCl NBT Parafn 4% paraformaldehyde 10X PCR buffer 0.1 M phosphate buffer 10 M anti-sense primer 10 M sense primer Proteinase K Streptavidin conjuguated to alkaline phosphatase 5 U/l Taq DNA polymerase 0.1 M TrisHCl buffer TrisHCl/CaCl2 buffer TrisHCl/NaCl buffer TrisHCl/NaCl/MgCl2 buffer DEPC water Sterile water Xylene or methyl cyclohexane 10.1.2.2 Preparation of samples
See Appendix B2.12. See Appendix B2.19. See Appendix B6.2.2.1. See Appendix B4.3.2. Use the one that is supplied with the enzyme. See Appendix B3.4.1. See Section 5.3.2. See Section 5.3.2. See Appendix B2.14.1. Or conjugated anti-biotin IgG See Appendix B2.21. See Appendix B3.7.2. See Appendix B3.7.5. See Appendix B3.7.6. See Appendix B1.2. See Appendix B1.1.
See Chapter 2.
10.1.2.2.1 CELL CULTURES Preparation of cells 6 10 cells/30 ml of medium, supplemented with 5 to 10% calf fetal serum and antibiotics, are cultivated on pretreated slides in culture boxes. The cells are conuent for: 2 days Washing 0.1 M phosphate buffer 2 5 min Fixation a. Incubate the sections: 4% paraformaldehyde 15 min in phosphate buffer
100 g/ml penicillin, 100 U/ml streptomycin See Appendix A3 or on Perkin-Elmer slides. Time variable according to the type of cell. This process requires great care, as the cells are delicate and risk becoming unstuck. It is important to pay attention to the xation time: insufcient xation leads to the diffusion of products, whereas excessively long xation hinders the penetration of the reagents.
282
10.1 b. Rinse: 0.1 M phosphate buffer Dehydration Ethanol 70, 95, 100 Drying Under a hood 10.1.2.2.2 FROZEN FIXED TISSUE Tissue preparation a. Fix: 4% paraformaldehyde b. Rinse: 0.1 M phosphate buffer 30% sucrose in 0.1 M phosphate buffer
Direct in Situ PCR
5 min 2 min per bath 3060 min Storage is possible at 20C.
26 h 4C 3 10 min <overnight
Depending on the size of the sample
Cryoprotection The samples must not oat to the surface of the solution, but rather sink to the bottom of the container. Freezing can also be carried out by contact with dry ice. This method is easier to reproduce. If possible For a few days, if the samples are mounted in O.C.T
c. Freeze: In liquid nitrogen vapor, or In isopentane cooled in liquid nitrogen. d. Store.
196C 160C 80C or 20C
Sections The sections are made by cryomicrotomy: Thickness 710 m Placed on PerkinElmer or silanated slides. Stored in hermetically 20C sealed boxes with a desiccant (e.g., silicagel).
See Appendix A3. Several months Note: Nonxed tissue sections must be xed with 4% paraformaldehyde before any pretreatment is carried out.
10.1.2.2.3 FIXED PARAFFIN-EMBEDDED TISSUE Preparation of tissue a. Fix: 4% paraformaldehyde 26 h 4C b. Rinse: 0.1 M phosphate buffer 2 15 min c. Dehydrate: Alcohol 70, 95, 100 12 h per bath
Depending on the size of the sample Or room temperature
283
Typical Protocols d. Impregnate: Xylene or methyl cyclohexane A solvent mixture, parafn (1:1 v/v) Parafn
12 h 12 h 56C 2 14 h 56C
Or the equivalent Toxic vapors
e. Embed in parafn. Sections The sections are made with a microtome. Thickness 710 m Placed on Perkin-Elmer slides
Placed on silanated slides Storage Storage in hermetically sealed boxes with a desiccant (e.g., silica gel)
Each slide can take two or three sections, which makes it possible to carry out different controls in the same experimental conditions (see Figure 5.4). See Appendix A3. Room temperature Several years For storage purposes, samples should preferably be embedded in parafn blocks. See Chapter 3. Condensation due to the warming of the slides should have disappeared.
rt
10.1.2.3
Pretreatments
10.1.2.3.1 CELL CULTURES AND FROZEN SECTIONS The return of the slides to 60 min room temperature rt Rehydration Ethanol 100, 95, 70 2 min per bath 9 NaCl 5 min Proteolytic digestion a. Incubate the slides: TrisHCl/CaCl2 5 min TrisHCl/CaCl2; 12 g/ml proteinase K 15 min 37C
b. Deactivate the enzyme.
2 min 95C
Proteinase K must be added extemporaneously to the buffer at 37C. Note: This digestion may cause the destruction of cells and produce background noise or a false-negative result due to the diffusion of the amplied product. Perform on a heating block. The proteolytic digestion can also be stopped by a TrisHCl/CaCl2 bath, with the addition of 2 mg/ml glycine.
c. Rinse: Sterile TrisHCl/CaCl2 d. Dehydrate: Ethanol 95, 100 Dry the sections under a hood. 284
2 5 min 2 min per bath 12 h
Pretreated slides can be stored at 20C.
10.1 Postxation a. Immediately after the proteolytic digestion step, the samples can be postxed to ensure the best adhesion of the section to the slide, and to stabilize the more fragile structures. 4% paraformaldehyde 5 min 0.1 M phosphate buffer 2 min 9 NaCl 2 min b. Dehydrate: Ethanol 95, 100 2 min per bath Dry the slides under a hood. 12 h 10.1.2.3.2 PARAFFIN-EMBEDDED FIXED TISSUE Dewaxing Xylene 2 15 min Alcohol 100 10 min Alcohol 100 5 min Alcohol 95 5 min Alcohol 70 5 min 9 NaCl 5 min Proteolytic digestion a. Incubate the slides: TrisHCl/CaCl2 5 min Proteinase K in 25 g/ml TrisHCl/CaCl2 15 min 37C An optional step
Direct in Situ PCR
Pretreated slides can be stored at 20C.
Or the equivalent
b. Deactivate the enzyme.
2 min 95C
Proteinase K must be added extemporaneously to the buffer at 37C. This concentration depends on the type of tissue. The risk of morphological destruction is much smaller for parafn-embedded tissue. Perform on a heating block. The proteolytic digestion can also be stopped at room temperature by a bath of TrisHCl/ CaCl2, with the addition of 2 mg/ml of glycine. Postxation is not necessary for parafnembedded tissue. Pretreated slides can be stored at 20C. See Chapter 5. The labels are essentially antigenic. During this amplication, nucleotides labeled with biotin are incorporated directly during the synthesis of the PCR products. 285
c. Rinse: TrisHCl/CaCl2 d. Dehydrate: Ethanol 95, 100 e. Dry the slides under a ventilated hood. 10.1.2.4 In situ amplication
2 5 min
2 min per bath 12 h
Reaction mixture for in situ amplication using labeled dNTP a. In a microtube placed in ice, prepare a reaction mixture containing:
Typical Protocols 10X PCR buffer 50 mM MgCl2 10 mM dTTP, dCTP, dGTP 0.4 mM biotin-14-dATP 10 l 3 l 2 l each 25 l 1 l 3 l Final concentration: 1X Final concentration: 1.5 mM Final concentration: 0.2 mM Final concentration: 0.1 mM Other labeled nucleotides (e.g., dUTPdigoxygenin, or dUTP-uorescein) can be used. Final concentration: 0.1 mM Final concentration: 0.3 M Labeled primers can be used (see Section 5.3.2.7). Final concentration: 0.3 M Final concentration: 0.1 U/l To a nal volume of 100 l Volume: 30 to 50 l, depending on the size of the sections To avoid evaporation during the hightemperature cycles During this amplication, the primers that were labeled during their synthesis by the coupling of antigenic molecules hybridize to one of the two strands of the denatured DNA and neosynthesize a complementary strand by taking it as a matrix. Final concentration: 1X Final concentration: 1.5 mM Final concentration: 0.2 mM 10 mM dTTP, dATP, dCTP, dGTP Final concentration: 1 M Final concentration: 1 M Final concentration: 0.1 U/l To a nal volume of 100 l Volume: 30 to 50 l. The slides are placed on a heating block at 95C, or the special Perkin-Elmer apparatus. To avoid evaporation during the hightemperature cycles
10 mM dATP 10 mM sense primer
10 M anti-sense primer 3 l 5 U/l Taq DNA polymerase 2.3 l DEPC water 46.7 l b. Place the reaction mixture on the sections. c. Cover the sections with ampli cover disks and cover clips (Perkin-Elmer) or Easyseal (Hybaid). d. Place the slides in a thermocycler. Reaction mixture for in situ amplication using labeled primers
a. In a microtube placed in ice, prepare the reaction mixture, with: 10X PCR buffer 10 l 50 mM MgCl2 3 l dNTP 2 l each 10 M sense primer 10 l 10 M anti-sense primer 10 l 5 U/l Taq DNA polymerase 2.3 l DEPC water 56.7 l b. Place the reaction mixture on the sections.
c. Cover the sections with ampli cover disks and cover clips (Perkin-Elmer), Easyseal (Hybaid); or use another method. d. Place the slides in a thermocycler. Amplication cycles a. Denature: Denaturing DNA 5 min 95C b. Preform the following cycles (20 cycles):
To eliminate protease Necessary to optimize the number of cycles with regard to the DNA in question, the tissue, and the primers
286
10.1 Denaturing Hybridization 1 min 95C 90 s 57C 90 s 74C 2 min 74C 10 s 30C 2 5 min 10 min 20C 5 min per bath
Direct in Situ PCR
Necessary to optimize the hybridization temperature with regard to the characteristics of the primers chosen
Extension c. Perform nal extension. d. Stop the reaction. e. Wash: 0.1 M TrisHCl buffer Postxation Cold ethanol 100 Rehydration Alcohol 95, 70
Possible to place the slides on hold if the thermocycler has been programmed at 4C After removing the clips, cover disks, or other To eliminate diffused products Fixation of the amplied products
10.1.2.5
Detection
Direct detection by conjugated streptavidin a. Incubate the slides in 5 min TrisHCl/NaCl buffer, with the additon of a blocking agent. b. Incubate: Streptavidin conjugated to 1h alkaline phosphatase diluted 37C to 1:500 in TrisHCl/NaCl buffer, with the addition of 1% ovalbumin c. Rinse: TrisHCl/NaCl buffer 3 10 min Direct detection by a conjugated antibody directed against the antigen in question a. Incubate the slides in 5 min TrisHCl/NaCl buffer added to a blocking agent. b. Incubate: Streptavidin conjugated 1h to alkaline phosphatase or rt peroxidase diluted to 1:100 to 1:500 in the blocking buffer, or Anti-digoxygenin conjugated 1h to alkaline phosphatase or rt peroxidase diluted to 1:100 to 1:500 in the blocking buffer, or
In a moisture chamber Possible to use streptavidin conjugated to peroxidase, but better results seem to be obtained with the alkalinephosphatase coupling
Depending on the label used
287
Typical Protocols Anti-uorescein, conjugated to 1h alkaline phosphatase or peroxidase rt diluted to 1:100 to 1:500 in the blocking buffer c. Rinse: TrisHCl/NaCl buffer 3 10 min Detection a. Incubate: TrisHCl/NaCl/MgCl2 5 min buffer, pH 9.6 b. Prepare the substrate: NBT BCIP TrisHCl/NaCl/MgCl2 buffer, pH 9.6 30 l 40 l 10 ml
See Appendix B3.7.6. DAB for detection with streptavidin or an IgG conjugated to peroxidase Final concentration: 0.3 mg/ml Final concentration: 0.2 mg/ml To inhibit endogenous phosphatase in some types of tissue, necessary to add levamisol (1 mM) to this preparation In the same way, possible to use hydrogen peroxide (H2O2) to inhibit endogenous peroxidase. Detection carried out in darkness under a microscope
c. Incubate until a signal 10 min2 h is obtained. rt d. Stop the reaction by washing 5 min in distilled water. Mounting the slides In an aqueous medium if the substrate is NBT/BCIP or Fast Red 10.1.2.6 Controls
Aquamount or Glycergel See Appendix B8.1.
Positive controls Tissue or cells containing the DNA being sought Negative controls Tissue or cells that are known not to contain the DNA being sought Reaction controls Omission of Taq DNA polymerase Omission of dNTP, whether labeled or not Omission of primers, whether labeled or not Detection-reaction controls Omission of the conjugate
The only way to nd out if the reaction has taken place normally in the case of negative results To conrm a specic amplication If the amplication step is omitted, no positive result Conrmation of the immunoenzymatic reaction No amplication step No detection step Revelation of endogenous enzymatic activity
288
10.2
Indirect in Situ PCR
10.2
INDIRECT IN SITU PCR
See Chapter 11, Figures 11.1 through 11.3.
Indirect in situ PCR, like direct in situ PCR, is essentially used to detect viral DNA or to identify foreign genes in cultured cells, frozen tissue sections, or xed, parafn-embedded tissue sections. Hybridization of the amplied product with the appropriate probes gives a further degree of specicity. The sample preparation and pretreatment steps are exactly the same as for direct PCR.
Gloves must be worn. All the products must be RNase-free, the solutions must be prepared in DEPC water (see Appendix B1.2), and the equipment must be sterilized (see Appendix A2). See Chapters 2 and 3.
289
Typical Protocols

10.2.2.1 Solutions See Appendix B2.2.
Ammonium acetate Anti-digoxygenin conjugated to alkaline phosphatase or peroxidase Anti-uorescein conjugated to alkaline phosphatase or peroxidase BCIP Blocking agent 10 mM dTTP, dCTP, dGTP dATP 10 mg/ml salmon sperm DNA Ethanol 100 (20C) Ethanol 70, 95, 100 Isopentane Liquid nitrogen 50 mM MgCl2 9 NaCl NBT Parafn 4% paraformaldehyde 10X PCR buffer 0.1 M phosphate buffer 10 M anti-sense primer 10 M sense primer Anti-sense probe Sense probe Proteinase K 10 mg/ml tRNA 20X SSC 0.1 M TrisHCl buffer TrisHCl/CaCl2 buffer TrisHCl/NaCl buffer TrisHCl/NaCl/MgCl2 buffer 5 U/l Taq DNA polymerase DEPC water Sterile water Xylene or methyl cyclohexane 10.2.2.2 10.2.2.3 Preparation of samples Pretreatments
See Appendix B6.2.2.1. See Appendix B6.2.1. dNTP See Appendix B2.8.
See Appendix B2.12. See Appendix B2.19. See Appendix B6.2.2.1. See Appendix B4.3.2. See Appendix B3.4.1. See Section 5.3.2. See Section 5.3.2. See Section 6.3. See Section 6.3. See Appendix B2.14.1. See Appendix B2.15. See Appendix B3.5. See Appendix B3.7.1. See Appendix B3.7.2. See Appendix B3.7.5. See Appendix B3.7.4. Supplied with the enzyme See Appendix B1.2. See Appendix B1.1.
See Section 10.1.2.2. See Section 10.1.2.3.
290
10.2 10.2.2.4 In situ amplication
Reaction mixture a. In a microtube placed in ice, prepare the reaction mixture: 10X PCR buffer 10 l 50 mM MgCl2 3 l 10 mM dNTP 2 l each 10 M sense primer 3 l 10 M anti-sense primer 3 l 5 U/l Taq DNA polymerase 2.3 l Sterile water 70.7 l b. Place the reaction mixture on the sections.
c. Cover the sections with ampli cover disks and clips (Perkin-Elmer), or Easyseal (Hybaid). d. Place the slides in a thermocycler. Amplication cycles a. Denature: Denaturing the DNA 5 min 95C b. Perform the following cycles: Denaturing 1 min 95C Hybridization 90 s 57C
Final concentration: 1X Final concentration: 1.5 mM Final concentration: 0.2 mM Final concentration: 0.3 M Final concentration: 0.3 M Final concentration: 0.1 U/l To a nal volume of 100 l Volume: 50 l On a heating block at 95C, or in the special Perkin-Elmer apparatus To avoid evaporation during the hightemperature cycles
20 cycles
The hybridization temperature must be optimized with regard to the characteristics of the primers chosen.
Extension c. Perform nal extension. d. Stop the reaction. e. Wash: TrisHCl buffer Fixation Cold ethanol 100 (20C) Drying Under a ventilated hood 10.2.2.5 10.2.2.5.1 Hybridization
90 s 74C 3 min 74C 10 s 30C 2 5 min 10 min 30 min1 h
The slides can be put on hold after the thermocycler has been programmed at 4C. Remove the clips and ampli cover disks, or other. Elimination of the diffused products Fixation of the amplied products
OF THE
LABELING PROBES BY EXTENSION 3 END
Hybridization after amplication is generally carried out with oligonucleotidic probes, and is thus the most commonly used type of labeling.
291
Typical Protocols Reaction mixture a. In a sterile microtube, prepare the reaction mixture: 100 pmol/l of oligonucleotides 1 l A 5X buffer specic to terminal 4 l transferase (TdT) 25 mM CoCl2 4 l 25 mmol Dig-11-dUTP 1 l 5 l 1 l
Final concentration: 5 pmol Final concentration: 1X Final concentration: 5 mM Final concentration: 1.25 mmol Or another labeled dUTP (see Section 5.3.1.3) To a nal volume of 20 l Final concentration: 1.25 U/l This enzyme is delicate, and must not be heated or vortexed, but shaken gently and, if necessary, centrifuged rapidly.
Sterile water 25 U/l terminal deoxy transferase
b. Incubate in a water bath.
1h 37C
c. Stop the action of the enzyme in ice. Precipitation a. Precipitate the probe by adding: tRNA 2 l Cold absolute ethanol 66 l Ammonium acetate 9 l b. Store. Overnight 20C c. Centrifuge. 30 min 14,000 g 4C d. Remove the supernatant. e. Dry the pellet in a vacuum jar or a Speedvac. Storage 20C
Store at 20C. See Appendix B2.2. Orient the tube in such a way to best observe the position of the centrifugation pellet. Eliminate all trace of alcohol. The probes are stored dry or solubilized in sterile distilled water.
10.2.2.5.2 HYBRIDIZATION Reaction medium a. In a sterile microtube placed in ice, the reaction mixture: 20X SSC Deionized formamide 50X Denhardts solution 10 mg/ml tRNA 10 mg/ml salmon sperm DNA Sense probe (1.25 pmol/l), labeled with digoxygenin Anti-sense probe (1.25 pmol/l), labeled with digoxygenin Sterile water
prepare 100 l 250 l 10 l 12.5 l 12.5 l 8 l 8 l 99 l Final concentration: 4X Final concentration: 50% Final concentration: 1X Final concentration: 250 g/ml Final concentration: 250 g/ml Final concentration: 20 pmol/ml
Final concentration: 20 pmol/ml To a nal volume of 500 l
292
10.2 b. Place the reaction mixture on the sections. c. Place sterile cover slides on the sections. d. Denature. 5 min 96C e. Cool immediately on ice. 5 min f. Incubate. Overnight 40C Volume: 30 to 50 l To avoid evaporation On a heating block
To stabilize the DNA in the form of single strands In a moisture chamber
Washing a. Remove the cover slides in a 4X SSC bath. b. Rinse: 2X SSC 2 30 min 0.5X SSC 30 min 10.2.2.6 Revelation
At room temperature At room temperature
Indirect detection by a conjugated antibody directed against the antigenic molecule in question a. Incubate the slides in TrisHCl/ 5 min NaCl buffer, with the addition of a blocking agent. b. Incubate with: Anti-biotin conjugated to alkaline 1h phosphatase or peroxidase rt Anti-digoxygenin conjugated to 1h alkaline phosphatase or peroxidase rt Anti-uorescein conjugated to 1h alkaline phosphatase or peroxidase rt c. Rinse: TrisHCl/NaCl buffer 3 10 min Detection a. Incubate: TrisHCl/NaCl/MgCl2 buffer, 5 min pH 9.6 b. Prepare the substrate: NBT BCIP TrisHCl/NaCl/MgCl2 buffer, pH 9.6 30 l 40 l 10 ml
Depending on the label Diluted to 1:100 to 1:500 in the blocking buffer Diluted to 1:100 to 1:500 in the blocking buffer Diluted to 1:100 to 1:500 in the blocking buffer
See Appendix B3.7.6 DAB in the case of detection with a streptavidin or an antibody conjugated to peroxidase Final concentration: 0.3 mg/ml Final concentration: 0.2 mg/ml To inhibit the endogenous phosphatases that are found in certain types of tissue, necessary to add levamisol (1 mM) to this preparation Likewise, hydrogen peroxide (H2O2) used to inhibit endogenous peroxidases Detection carried out in darkness under a microscope
c. Incubate until a signal is obtained. d. Stop the reaction by washing in distilled water.
10 min2 h rt 5 min
293
Typical Protocols Mounting the slides n an aqueous medium, if the substrate is NBT/BCIP or Fast Red n a synthetic medium, after dehydration in alcohol and dipping in solvents, if the substrate is DAB 10.2.2.7 Controls
Aquamount or Glycergel (see Appendix B8.1) Entellan or Eukitt (see Appendix B8.2)
See Chapter 9. The only way to nd out if the reaction has taken place normally in the case of negative results To conrm a specic amplication If the amplication step omitted, no positive result No amplication step Conrmation of the immunoenzymatic reaction Negative result Absence of phosphatases or endogenous peroxidases
Positive controls Tissue or cells containing the DNA being sought Negative controls Tissue or cells that are known not to contain the DNA being sought Reaction controls Omission of Taq DNA polymerase Omission of primers Omission of the labeled dNTP Detection controls Omission of probes Omission of antibodies
10.3
INDIRECT IN SITU RT-PCR

This is the only way to visualize mRNA that is not strongly expressed in a particular type of cell. Thus, the detection of a weakly expressed gene, an abnormal gene, or an RNA virus is a potential application of in situ RT-PCR. The steps involved in preparing the samples, and the pretreatments, are exactly the same as for direct PCR.
Gloves must be worn. All the products must be RNase-free, the solutions must be prepared in DEPC water (see Appendix B1.2), and the equipment must be sterilized (see Appendix A2). In the case of RNA, which breaks down very easily, these conditions must be even more strictly respected. See Sections 10.1.2.2 and 10.1.2.3.
294
10.3
Indirect in Situ RT-PCR

10.3.2.1 Solutions
Anti-biotin conjugated to alkaline phosphatase or peroxidase Anti-digoxygenin conjugated to alkaline phosphatase or peroxidase Anti-uorescein, conjugated to alkaline phosphatase or peroxidase BCIP Blocking agent 2 mM CaCl2
See Appendix B6.2.2.1. See Appendix B6.2.1. See Appendix B2.3.2. 295
Typical Protocols Diaminobenzidine (DAB) 10 mg/ml salmon sperm DNA DNase 0.1 M DTT 10 mM dTTP, dCTP, dGTP, dATP Ethanol 100 (20C) Ethanol 70, 95, 100 Deionized formamide 50X Denhardts solution Isopentane Liquid nitrogen 6 mM; 50 mM MgCl2 9 NaCl NBT Parafn 4% paraformaldehyde 10X PCR buffer 0.1 M phosphate buffer Proteinase K 10 M anti-sense primer 10 M sense primer Anti-sense probe Sense probe 200 U/l reverse transcriptase 10 mg/ml tRNA 40 U/l RNasin 5X RT buffer 20X SSC Streptavidin conjugated to alkaline phosphatase 5 U/l Taq DNA polymerase TrisHCl buffer TrisHCl/CaCl2 buffer TrisHCl/NaCl buffer TrisHCl/NaCl/MgCl2 buffer DEPC water Sterile water Xylene or methyl cyclohexane 10.3.2.2 10.3.2.3 10.3.2.4 Preparation of samples Pretreatments DNase treatment See Appendix B6.2.2. See Appendix B2.8. See Appendix B2.9. See Appendix B2.7. dNTP
See Appendix B2.4. See Appendix B2.5. See Appendix B2.12. See Appendix B2.19. See Appendix B6.2.2.1. See Appendix B4.3.2. Supplied with the enzyme See Appendix B3.4.1. See Appendix B2.14.1. See Section 5.3.2. See Section 5.3.2. See Section 6.3. See Section 6.3.
See Appendix B2.15. Supplied with the enzyme See Appendix B3.5.
See Appendix B3.7.1. See Appendix B3.7.2. See Appendix B3.7.5. See Appendix B3.7.4. See Appendix B1.2. See Appendix B1.1.
See Section 10.1.2.2. See Section 10.1.2.3. Optional step if the primers, and especially the probes, are specic for the sequence to be amplied. The DNase must be of RNase-free quality.
a. Prepare the DNase solution: 40 mM TrisHCl, pH 7.4 6 mM MgCl2 2 mM CaCl2 296
10.3 1 to 100 U/ml of DNase in the nal volume
For cells and tissue that are particularly rich in ribonuclease, 1000 U/ml RNasin and 1 mM dithiothreitol (DTT) should be added to the DNase solution. Time depends on the concentration.
b. Cover the sections with 20 to 30 l of this solution. c. Incubate in a moisture chamber. 112 h 37C d. Rinse the sections in the DNase dilution buffer. e. Deactivate the enzyme. 2 min 95C f. Rinse the slides in DEPC water. 10.3.2.5 Reverse transcription
On a heating block
Reaction mixture a. In a sterile microtube, prepare the reaction mixture: 5X RT buffer 20 l 0.1 M DTT 10 l 10 mM dNTPs 5 l 40 U/l RNasin 2.5 l 10 M anti-sense primer 10 l DEPC water 47.5 l 200 U/l reverse transcriptase 5 l b. Place the reaction mixture on the sections. c. Cover the sections with ampli cover disks and cover clips (Perkin-Elmer), Easyseal (Hybaid), or simply sealed cover slides. d. Place the slides 60 min in the thermocycler. 40C e. Deactivate the enzyme. 2 min 94C Washing 0.1 M phosphate buffer 5 min 9 NaCl 2 min Dehydration Ethanol 95, 100 Drying Under a ventilated hood
Final concentration: 1X Final concentration: 10 mM Final concentration: 0.5 mM Final concentration: 1 U/l Final concentration: 1 M To a nal volume of 100 l Final concentration: 10 U/l Volume: 30 to 50 l To avoid evaporation See Section 4.4.2
The temperature at which reverse transcriptase activity is optimal The temperature, at which the enzyme is destroyed To avoid phosphate precipitation (whitish streaks)
2 min per bath Traces of alcohol could precipitate the polymerase.
10.3.2.6
In situ amplication
Reaction mixture In a sterile microtube placed in ice, prepare the reaction mixture: 10X PCR buffer 10 l
Final concentration: 1X 297
Typical Protocols 25 mM MgCl2 Mixture of 10 mM dNTP 10 M sense primer 10 M anti-sense primer DEPC water The hot start 6 l 5 l 10 l 10 l 55 l Final concentration: 1.5 mM Final concentration: 0.2 mM Final concentration: 1 M Final concentration: 1 M To a nal volume of 96 l The specicity of primer matching must be ensured. There is a high risk of nonspecic hybridization at low temperature. Final concentration: 0.2 U/l Volume: 50 l. The sections are placed on a heating block at 95C, or the special PerkinElmer apparatus. To avoid evaporation during the hightemperature cycles
5 min 82C b. Add 5 U/l Taq DNA polymerase. 4 l c. Place the reaction mixture on the sections.
a. Incubate the mixture.
d. Cover the sections with the ampli cover disks and clips (Perkin-Elmer), Easyseal (Hybaid), or simply sealed cover slides. e. Place the slides in a thermocycler. Amplication cycles a. Program 25 amplication cycles: Denaturing 1 min 94C Hybridization 90 s 45C Extension b. Perform nal extension. c. Stop the reaction. d. Wash: 0.1 M phosphate buffer Postxation 4% paraformaldehyde 0.1 M phosphate buffer 9 NaCl Dehydration Ethanol 95, 100 Drying Under a ventilated hood 10.3.2.7 Hybridization 2 5 min 15 min 5 min 2 min 2 min per bath 30 min1 h 90 s 72C 5 min 72C 10 s 30C
The hybridization temperature must be optimized according to the characteristics of the primers.
The slides can be stored at 4C in the thermocycler. Remove the clips and the ampli-cover disks, or other. Eliminate diffused products. Fix the amplied products.
Reaction mixture a. In a sterile microtube placed in ice, prepare the reaction mixture: 20X SSC 100 l 298
Final concentration: 4X
10.3 Deionized formamide 250 l 50X Denhardts solution 10 l 10 mg/ml tRNA 12.5 l 10 mg/ml salmon sperm DNA 12.5 l Sense probe (1.25 pmol/l) 8 l labeled with digoxygenin Anti-sense probe (1.25 pmol/l) 8 l labeled with digoxygenin Sterile water 99 l b. Place the reaction mixture on the slides. c. Place sterile coverslips on the sections. d. Denature. 5 min 96C e. Cool immediately on ice. 5 min f. Incubate. Overnight 40C
Final concentration: 50% Final concentration: 1X Final concentration: 250 g/ml Final concentration: 250 g/ml Final concentration: 20 pmol/ml Or another label (e.g., biotin uorescein) Final concentration: 20 pmol/ml Or another label (e.g., biotin uorescein) To a nal volume of 500 l Volume: 30 to 50 l To avoid evaporation On a heating block
To stabilize the DNA in the form of single strands In a moisture chamber containing 5X SSC
Washing a. Detach the coverslips in a 4X SSC bath. b. Rinse: 2X SSC 2 30 min 0.5X SSC 30 min 10.3.2.8 Revelation
At room temperature At room temperature
Direct detection by a conjugated antibody directed against the antigenic molecule being used a. Incubate the slides in TrisHCl/ 5 min NaCl, added to a blocking agent. b. Incubate with: Anti-digoxygenin conjugated to 1h alkaline phosphatase or peroxidase rt
Ovalbumin, goat serum Depending on the label Diluted to 1:100 to 1:500 in the blocking buffer Or another system, depending on the label (e.g., anti-biotin or anti-uorescein)
c. Rinse: TrisHCl/NaCl buffer 3 10 min Phosphatase alkaline detection a. Incubate: TrisHCl/NaCl/MgCl2 buffer, 5 min pH 9.6 b. Prepare the substrate extemporaneously: 30 l 40 l 10 ml
NBT BCIP TrisHCl/NaCl/MgCl2 buffer, pH 9.6
DAB in the case of detection with an antibody conjugated with peroxidase (see Chapter B6.2.2.2) Final concentration: 0.3 mg/ml Final concentration: 0.2 mg/ml To inhibit the phosphatases that are endogenous to certain types of tissue, necessary to add levamisol (1 mM) to this preparation In the same way, hydrogen peroxide (H2O2) used to inhibit endogenous peroxidases 299
Typical Protocols c. Incubate until a signal is 10 min2 h obtained. rt d. Stop the reaction by washing 5 min in TrisHCl buffer. Mounting the sections In an aqueous medium if the substrate is NBT/BCIP or Fast Red In a synthetic medium after dehydration in alcohol, and baths of solvent if the substrate is DAB 10.3.2.9 Controls Detection carried out in darkness under a microscope
Aquamount or Glycergel (see Appendix B8.1) Entellan, Eukitt (see Appendix B8.2)
See Chapter 9. The only way to nd out if the reaction has taken place normally in the case of negative results To conrm a specic amplication
Positive controls Tissue or cells containing the mRNA being sought Negative controls Tissue or cells known not to contain the mRNA being sought Reaction controls Omission of reverse transcriptase Omission of Taq DNA polymerase Omission of primers Omission of labeled dNTP Detection controls Omission of probes
If the amplication step omitted, no positive result No amplication step Conrmation of the immunoenzymatic reaction Possible that a weak signal may persist even though there is no amplication step; which results from the hybridization of cDNA synthesized during the RT step Negative results; the demonstration of endogenous enzymatic activity
Omission of antibodies
10.4
DIRECT IN SITU RT-PCR SPECIAL CASE: CELL SUSPENSION

Gloves must be worn. All the products must be RNase-free, solutions must be prepared in DEPC water (see Appendix B1.2), and the equipment must be sterilized (see Appendix A.2).
This protocol is applicable to all suspended cells, whether derived from cultures, biological uids, or even the mechanical or enzymatic dissociation of tissue. The isolation protocols are different in each case, and we would simply recall
300
10.4 that the integrity of the cells must be conserved, as RT and PCR reactions take place in the cytoplasm. Here we will give the xation and permeabilization procedures that must be carried out before RT-PCR.
Direct in Situ RT-PCR
In the case of RNA, which breaks down very easily, these conditions must be even more strictly respected.
Direct in situ RT-PCR using ourescent primers
301
Typical Protocols Direct in situ RT-PCR using biotinylated primers
10.4.2 Typical Protocol

10.4.2.1 Solutions
Anti-biotin conjugated to alkaline phosphatase or peroxidase Anti-digoxygenin conjugated to alkaline phosphatase or peroxidase Anti-uorescein conjugated to alkaline phosphatase or peroxidase BCIP 302
See Appendix B6.2.2.1
10.4 0.4 mM biotin-14-dATP Blocking agent 2 mM CaCl2 50X Denhardts solution Diaminobenzidine (DAB) 10 mg/ml salmon sperm DNA 0.1 M DTT 10 mM dTTP, dCTP, dGTP dATP dUTP conjugated to digoxygenin dUTP conjugated to uorescein Ethanol 100 (20C) Ethanol 70, 95, 100 10% formol Hmaluns solution Igepal CA-630 Isopentane 50 mM; 6 mM MgCl2 9 NaCl NBT Parafn 4% paraformaldehyde PBS PBS-glycerol 0.1 M phosphate buffer 10X PCR buffer 10 M anti-sense primer 10 M sense primer Anti-sense probe Sense probe Proteinase K 20X SSC 200 U/l reverse transcriptase 10 mg/ml tRNA 40 U/l RNasin 5X RT buffer Streptavidin conjugated to alkaline phosphatase 5 U/l Taq DNA polymerase 40 mM; 100 mM TrisHCl buffer TrisHCl/CaCl2 buffer TrisHCl/NaCl buffer TrisHCl/NaCl/MgCl2 buffer DEPC water Sterile water Xylene or methyl cyclohexane 10.4.2.2 Fixation
See Appendix B6.2.1. See Appendix B2.3. See Appendix B2.5. See Appendix B6.2.2.2. See Appendix B2.8. See Appendix B2.7.
See Appendix B4.1. See Appendix B7.1.3. See Appendix B2.12. See Appendix B2.19. See Appendix B6.2.2.1. See Appendix B4.3.2. See Appendix B4.3.4.3. See Appendix B8.1. See Appendix B3.4.1. Supplied with the enzyme See Section 5.3.2. See Section 5.3.2. See Section 6.3. See Section 6.3. See Appendix B2.14.1. See Appendix B3.5.
See Appendix B2.15. Supplied with the enzyme
See Appendix B3.7.1. See Appendix B3.7.2. See Appendix B3.7.5. See Appendix B3.7.4. See Appendix B1.2.
The cells are rinsed in PBS before being centrifuged and separated into aliquots of a minimum 7 of 10 cells in sterile microtubes.
The minimum number of cells is important, since subsequent washes and operations lead to the loss of a large number of cells. 303
Typical Protocols 3 min 1500 g b. Remove the supernatant and put the suspended pellet in 1 ml of 4% paraformaldehyde or 10% formol, and 0.15 M NaCl. c. Incubate in ice, shaking frequently. 1h d. Centrifuge. 3 min 1500 g e. Suspend the pellet in PBS. 4C f. Centrifuge. 3 min 1500 g Perform this washing step three times. 10.4.2.3 Pretreatments Nonionic detergent whose chemical composition is similar to that of Nonidet P 40, which is no longer available Possible to use other detergents Melting ice Important to use a refrigerated centrifuge a. Centrifuge.
Use a Pasteur-type pipette to suspend the pellet. See Appendix B2.19. It is important to use a refrigerated centrifuge.
a. Suspend the pellet in Igepal CA-630. 0.5%
b. Incubate, shaking frequently. c. Centrifuge. d. Suspend the pellet in PBS.
1h 0C 3 min 1500 g 4C
Perform this washing step three times. e. After the last centrifugation, count the cells, make sure that they are not clustered, and 6 adjust the concentration to 2 10 cells/ml. 10.4.2.4 Reverse transcription If the cells are not to be used immediately, the aliquots can be frozen and stored at 80C.
Reaction mixture a. In a sterile microtube, prepare the reaction mixture: 5X RT buffer 10 l 0.1 M DTT 5 l 10 mM dNTPs 2.5 l 40 U/l RNasin 2.5 l 10 M anti-sense primer 5 l DEPC water 22.5 l b. Suspend the pretreated cell pellet in the calculated quantity of distilled water. c. Add the reaction mixture, and homogenize by careful pipetting. d. Add 200 U/l reverse transcriptase, 2.5 l and mix once more by careful pipetting. e. Incubate. 1h 37C or, 1h 42C 304
Final concentration: 1X Final concentration: 10 mM Final concentration: 0.5 mM Final concentration: 1 U/l Final concentration: 1 M To a nal volume of 47.5 l
Final concentration: 10 U/l Final volume: 50 l If MMLV is the reverse transcriptase used If AMV is the reverse transcriptase used
10.4 f. Deactivate the enzyme. Washing a. Centrifuge. 2 min 94C 3 min 1500 g
At this temperature, the enzyme is destroyed. Although the pellet is generally quite visible, it is a good idea always to place the tube in the same position in the centrifuge.
b. Remove all the supernatant, and suspend the pellet in 200 l PBS. c. Centrifuge once more. 2 min 1500 g d. Remove the supernatant, and suspend the pellet in 100 l PBS.
The cells can be used directly in PCR. They can also be frozen and stored at 80C in aliquots of 10 l.
10.4.2.5
Amplication with labeled primers The use of two uorochromes makes it possible to detect two different mRNA sequences. This codetection can be an advantage for a number of applications.
10.4.2.5.1 FLUORESCENT PRIMERS For some applications, amplication can be carried out in the presence of labeled primers, notably a uorochrome for the direct visualization of the amplied product. Although not recommended, this method makes possible the detection of very small numbers of positive cells by ow cytometry. Reaction mixture a. In a sterile microtube, prepare the reaction mixture: 10X PCR buffer 5 l 25 mM MgCl2 3 l Mixture of 10 mM dNTP 2.5 l 10 M labeled sense primer 5 l
10 M labeled anti-sense primer 5 l Sterile water 27.5 l b. Add the reaction mixture to the cell pellet, and homogenize by careful pipetting. The hot start
Final concentration: 1X Final concentration: 1.5 mM Final concentration: 0.2 mM Final concentration: 1 M Only one labeled primer can be used, but to this extent the effectiveness of the amplication will decline. Final concentration: 1 M To a nal volume of 48 l The specicity of primer matching must be ensured. There is a high risk of nonspecic hybridization at low temperature. Final concentration: 0.2 U/l Final volume: 50 l To facilitate the washing steps, it is best not to add mineral oil.
a. Incubate the mixture. b. Add 5 U/l Taq DNA polymerase, and check that the tube is closed.
5 min 82C 2 l
305
Typical Protocols Amplication cycles a. Program 25 amplication cycles: Denaturing Hybridization
1 min 94C 90 s 45C 90 s 72C 5 min 72C 10 s 30C 3 min 1500 g
The hybridization temperature must be optimized in keeping with the characteristics of the primers used.
Extension b. Perform nal extension. c. Stop the reaction. Washing a. Centrifuge.
The slides can be put on hold after the thermocycler has been programmed at 4C. If mineral oil has been used, it is important, to avoid contamination, to extract the cells from the liquid phase and suspend them in 200 l PBS before centrifuging.
b. Remove all the supernatant and suspend the pellet in 200 l PBS. c. Centrifuge once more. 2 min 1500 g d. Remove the supernatant and suspend the pellet in 100 l PBS-glycerol (1:1 v/v). e. Place a 50 l drop on a slide, and cover it with a cover slide. f. Remove the supernatant and suspend the pellet in 0.5 ml PBS. 10.4.2.5.2 BIOTINYLATED PRIMERS
For examination by uorescence microscope or confocal microscope For ow cytometry studies
Reaction mixture a. In a sterile microtube placed in ice, prepare a reaction mixture containing: PCR 10X buffer 5 l 25 mM MgCl2 3 l 10 mM dNTP mixture 2.5 l 10 M labeled sense primer 5 l
10 M anti-sense primer 5 l Sterile water 27.5 l b. Add the reaction mixture to the cell pellet, and homogenize by careful pipetting. The hot start
Final concentration: 1X Final concentration: 1.5 mM Final concentration: 0.2 mM Final concentration: 1 M Only one labeled primer can be used, but to this extent the effectiveness of the amplication will decline. Final concentration: 1 M To a nal volume of 48 l The specicity of primer matching must be ensured. There is a high risk of nonspecic hybridization at low temperature.
306
10.4 a. Incubate the mixture. b. Add 5 U/l Taq DNA polymerase, and check that the tube is closed. Amplication cycles a. Program 25 amplication cycles: Denaturing Hybridization 5 min 82C 2 l
Final concentration: 0.2 U/l Final volume: 50 l To facilitate the washing steps, it is best not to add mineral oil.
1 min 94C 90 s 45C 90 s 72C 5 min 72C 10 s 30C 3 min 1500 g
Extension b. Perform nal extension. c. Stop the reaction. Washing a. Centrifuge
If mineral oil has been used, it is important, to avoid contamination, to extract the cells from the liquid phase and suspend them in 200 l PBS before centrifuging.
b. Remove all the supernatant and suspend the pellet in 200 l PBS. c. Centrifuge once more. 2 min 1500 g d. Remove all the supernatant and suspend the pellet in 200 l PBS. Peroxidase detection a. Place 50 l of the suspension on a pretreated slide, and spread it carefully to obtain a smear. b. Leave to dry. 30 min c. Incubate in a moisture chamber 1h with 200 l of streptavidin rt conjugated with the peroxidase diluted to 1:100 in TrisHCl/NaCl. d. Rinse in TrisHCl/NaCl in 5 min a tray, with light shaking. e. Incubate with 200 l of DAB 5 min prepared extemporaneously. f. Rinse in TrisHCl/NaCl in a tray, 5 min with light shaking. g. A counterstaining can be carried out 30 s by dipping the slides in Hemaluns solution.
If the cells are to adhere correctly, it is essential that the slides be pretreated (see Appendix A3). Streptavidin can be conjugated to alkaline phosphatase Anti-biotin conjugated can also be used.
Keep an eye on the detection procedure.
Check that this counterstaining does not interfere with the interpretation of the signal. See Appendix B7.1.3.
307
Typical Protocols h. Rinse rapidly in running water, 2 min and dehydrate in alcohol baths per bath of increasing strength, followed by a bath of methyl cyclohexane. i. Mount the slide in Entellan or DPX.
Observation is by conventional microscopy. The blue nuclei contrast with the brown cytoplasmic staining of the amplied cDNA.
10.4.2.6
Controls The only way to nd out if the reaction has taken place normally in the case of negative results To conrm a specic amplication Negative control; only the hybridization of RNA can give rise to a signal If the amplication step omitted, no positive result should be obtained No amplication step If the amplied products have diffused, a band will be clearly visible on the gel.
Positive controls Cells that contain the mRNA being sought. Negative controls Cells that are known not to contain the mRNA being sought Reaction controls Omission of reverse transcriptase Omission of Taq DNA polymerase Omission of labeled primers Diffusion control 10 l of the reaction mixture is removed after the amplication and subjected to electrophoresis.
10.5
INDIRECT IN SITU RT-PCR ON VEGETABLE TISSUE USING FLOATING VIBRATOME SECTIONS

See Chapter 11, Figure 11.11.
For vegetable tissue the method of choice for visualizing weakly expressed mRNA is an adaptation of the in situ RT-PCR on oating vibratome sections, which can be applied to vegetable tissue with only small modications. The difculties concern the tissue structures. In the example given here, the use of thick, xed tissue sections made on a vibratome ensures a high detection threshold in well-conserved tissue structure.
Gloves must be worn. All the products must be RNase-free, the solutions must be prepared in DEPC water (see Appendix B1.2), and all equipment must be sterilized (see Appendix A2). In the case of RNA, which breaks down very easily, these conditions must be even more strictly respected.
308
10.5
Indirect in Situ RT-PCR on Vegetable Tissue Using Floating Vibratome Sections
309
Typical Protocols

10.5.2.1 Solutions RNase free Low melting temperature See Appendix B6.2.1. Or anti-uorescein conjugated to alkaline phosphatase or peroxidase See Appendix B2.15. See Appendix B2.5. See Appendix B2.8. See Appendix B2.7. dNTP See Appendix B2.12. See Appendix B2.19. See Appendix B4.3.2. See Appendix B3.4.3. See Appendix B3.4.1. See Section 5.3.2. See Section 5.3.2. See Section 6.3. See Section 6.3. See Appendix B2.14.1. MMLV
5% agarose Blocking agent Anti-digoxygenin conjugated to alkaline phosphatase or peroxidase 10 mg/ml tRNA 50X Denhardts solution 10 mg/ml salmon sperm DNA 0.1 M DTT 10 mM dTTP, dCTP, dGTP, dATP 6 mM; 50 mM MgCl2 9 NaCl 4% paraformaldehyde PBS 10X PCR buffer 0.1 M phosphate buffer 10 M anti-sense primer 10 M sense primer Anti-sense probe Sense probe Proteinase K 200 U/l reverse transcriptase 10 mg/ml RNase A 40 U/l RNasin 5X RT buffer 20X SSC 5 U/l Taq DNA polymerase 0.1 M TrisHCl buffer 40 mM TrisHCl buffer TrisHCl/CaCl2 buffer TrisHCl/NaCl buffer TrisHCl/NaCl/MgCl2 buffer DEPC water Sterile water 10.5.2.2 Fixation
See Appendix B3.5. See Appendix B3.7.1. See Appendix B3.7.1. See Appendix B3.7.2. See Appendix B3.7.5. See Appendix B3.7.4. See Appendix B1.2. See Appendix B1.1.
a. Fix the sample in 4% 12 h paraformaldehyde in 100 mM phosphate buffer, pH 7.2 using the conditions mentioned above. b. Rinse in phosphate buffer. 2 60 min
According to the size of the sample Addition of a low percentage of glutaraldehyde (e.g., 0.1%) (see Section 2.1.3.3)
310
10.5 10.5.2.3
Sections RNase free Use cyanolit-type glue. The area of the section must be large enough not to hamper subsequent procedures. All the different RT, PCR, and hybridization steps are carried out on oating sections in microtubes.
a. Embed in 5% agarose. b. Cool on ice. c. Adhere the sample to the object-holder of the vibratome. d. Immerse the sample in 2X SSC to obtain 80to 100-m-thick oating sections. e. Remove the agarose mechanically by hand. f. Place two to ve sections in TrisHCl/NaCl/ CaCl2 buffer in microtubes.
10.5.2.4
Pretreatments 5 min 37C 2 g/ml 15 min 37C rt
a. Incubate the sections in TrisHCl/NaCl/CaCl2 buffer. b. Add proteinase K.
c. Rinse in TrisHCl/NaCl/CaCl2 buffer. d. Transfer the sections to other microtubes containing 0.1 M phosphate buffer, pH 7.4. 10.5.2.5 Reverse transcription
Reaction mixture a. In a sterile microtube, prepare the reaction mixture: 5X RT buffer 20 l 0.1 M DTT 10 l 10 mM dNTPs 5 l 40 U/l RNasin 2.5 l 10 M anti-sense primer 10 l Sterile water 47.5 l b. Replace the phosphate buffer with the reaction mixture, and pipette carefully to suspend the sections. c. Add 200 U/l reverse 5 l transcriptase. d. Incubate in a thermocycler. 1h 37C e. Deactivate the enzyme. 2 min 94C Washing The sections are rinsed in 5 min phosphate buffer.
Final concentration: 1X Final concentration: 10 mM Final concentration: 0.5 mM Final concentration: 1 U/l Final concentration: 1 M To a nal volume of 95 l
Final concentration: 10 U/l Final volume: 100 l If MMLV is the reverse transcriptase used The temperature at which the enzyme is destroyed
311
Typical Protocols 10.5.2.6 Amplication
Reaction mixture a. In a sterile microtube placed in ice, prepare the reaction mixture: 10X PCR buffer 10 l 25 mM MgCl2 6 l 10 mM dNTP mixture 5 l 10 M sense primer 10 l 10 M anti-sense primer 10 l Sterile water 55 l b. Replace the phosphate buffer with the reaction mixture, and suspend the sections by careful pipetting. The hot start
Final concentration: 1X Final concentration: 1.5 mM Final concentration: 0.2 mM Final concentration: 1 M Final concentration: 1 M To a nal volume of 96 l
The specicity of primer matching must be ensured. There is a high risk of nonspecic hybridization at low temperature. Final concentration: 0.2 U/l Final volume: 100 l To facilitate the subsequent washing steps, it is best not to add mineral oil. The number of cycles must be optimized for each operation.
a. Incubate. b. Add 5 U/l Taq DNA polymerase, and check that the tube is closed. Amplication cycles a. Program 10 amplication cycles: Denaturing Hybridization
5 min 82C 4 l
1 min 94C 90 s 50C 90 s 72C 5 min 72C 10 s 30C
Extension b. Perform nal extension. c. Stop the reaction.
The sections can be stored at 4C in a thermocycler.
Washing a. Remove all the reaction medium. b. Suspend the sections in 2 10 min phosphate buffer. Postxation 4% paraformaldehyde in 10 min 2X SSC buffer Rinsing in 2X SSC 3 5 min
To x the hybrids that have been formed, and to stabilize the tissue and cell structures
312
10.5 10.5.2.7
Hybridization See Chapter 6. Sense and anti-sense probes labeled by PCR Fluorescein or biotin.
The amplied products are detected by hybridization, using two cDNA probes labeled with digoxygenin. Reaction mixture a. In a sterile microtube placed in ice, prepare the reaction mixture: 20X SSC 100 l Deionized formamide 250 l 50X Denhardts solution 10 l 10 mg/ml tRNA 12.5 l 10 mg/ml salmon sperm DNA 12.5 l Labeled sense probe 8 l Labeled anti-sense probe 8 l
Sterile water 99 l b. Replace the phosphate buffer with the reaction mixture. c. Suspend the sections. d. Denature. 5 min 95C e. Cool immediately on ice. 5 min f. Incubate. Overnight 5560C Washing 2X SSC
Final concentration: 4X Final concentration: 50% Final concentration: 1X Final concentration: 250 g/ml Final concentration: 250 g/ml Final concentration: 0.01 to 0.1 g/ml of hybridization buffer Final concentration: 0.01 to 0.1 g/ml of hybridization buffer To a nal volume of 500 l
On a heating block To stabilize the DNA in single-strand form In the thermocycler .The hybridization temperature must take into account the characteristics of the probes. See Chapter 6, Figure 6.4. It is always possible to increase or decrease the concentration of the SSC buffer and the washing time in line with the results obtained.
30 min rt 30 min rt 30 min 37C 4560 min 37C 3 10 min 30 min 60C 30 min rt 30 min rt
1X SSC TE/NaCl RNase A treatment 100 g/ml in TE/NaCl TE/NaCl 1X SSC 0.5X SSC 0.1X SSC
The duration and temperature can be increased to reduce background. The temperature can be increased to reduce background
313
Typical Protocols 10.5.2.8 Immunocytological detection
Direct detection by a conjugated antibody directed against the antigenic molecule used a. Incubate with: Mouse anti-label monoclonal >2 h antibody conjugated to alkaline rt phosphatase at a dilution of 1:50 in TrisHCl/NaCl buffer added to a blocking agent and a detergent b. Rinse: In the same buffer, but 2 30 min without detergent Phosphatase alkaline detection a. Incubate: TrisHCl/NaCl/MgCl2 buffer, 5 min pH 9.6 b. Prepare the substrate extemporaneously: 30 l 40 l 10 ml
According to the label On drops of reagent, with the section placed on the liquid Digoxygenin, uorescein, or biotin Example: 0.5% ovalbumin Example: 0.05% Tween 20 Or more
NBT BCIP TrisHCl/NaCl/MgCl2 buffer, pH 9.6
c. Incubate until a signal 10 min2 h is obtained. rt d. Stop the reaction by washing 15 min in TrisHCl buffer. Mounting the sections In an aqueous medium if the substrate is NBT/BCIP or Fast Red In a synthetic medium after dehydration in alcohol, and baths of solvent if the substrate is DAB 10.5.2.9 Observations
DAB in the case of detection with an antibody conjugated with peroxidase (see Chapter B6.2.2.2). Final concentration: 0.3 mg/ml Final concentration: 0.2 mg/ml To inhibit the phosphatases that are endogenous to certain types of tissue, necessary to add levamisol (1 mM) to this preparation In the same way, hydrogen peroxide (H2O2) used to inhibit endogenous peroxidases Detection carried out in darkness under a microscope
Aquamount or Glycergel (see Appendix B8.1) Entellan, Eukitt (see Appendix B8.2)
The sections are observed by light microscopy.
314
10.6 10.5.2.10 Controls
Indirect in Situ RT-PCR Using Electron Microscopy
Positive controls Tissue containing the mRNA being sought Negative controls Tissue that is known not to contain the mRNA being sought Reaction controls Omission of reverse transcriptase, or treatment of tissue with RNase Omission of Taq DNA polymerase Omission of primers
This is the only way to nd out if the reaction has taken place normally in the case of negative results. To conrm a specic amplication Control of the reverse transcription If the amplication step omitted, no positive result Possible that a weak signal may, however, appear, due to the endogenous reparative power of Taq DNA polymerase Detection control If the amplied products have diffused, a band will be clearly visible on the gel.
Omission of the antibody Diffusion control 10 l of the reaction medium is removed after amplication, and subjected to electrophoresis.
10.6
INDIRECT IN SITU RT-PCR USING ELECTRON MICROSCOPY

In situ RT-PCR using electron microscopy is currently one of the methods of choice for visualizing mRNA that is weakly expressed in a precise subcellular compartment. More than for any other approach, this identication necessitates high morphological quality, and the protocols must take account of this necessity, as in the example given here, where the use of thick, xed tissue sections made on a vibratome ensures a high detection threshold in a wellconserved cell structure.
Gloves must be worn. All the products must be RNase-free, the solutions must be prepared in DEPC water (see Appendix B1.2), and all equipment must be sterilized (see Appendix A2). In the case of RNA, which breaks down very easily, these conditions must be even more strictly respected.
315
Typical Protocols

10.6.2.1 Solutions See Appendix B7.2.1.2. See Appendix B2.8. See Appendix B6.2.1.
5% aqueous uranyl acetate 10 mg/ml salmon sperm DNA Blocking agent Anti-species antibody conjugated to colloidal gold Anti-digoxygenin conjugated to alkaline phosphatase or peroxidase 316
10.6 Anti-uorescein conjugated to alkaline phosphatase or peroxidase 0.4 mM biotin-14-dATP 2 mM CaCl2 50X Denhardts solution Digoxygenin-11-dATP 0.1 M DTT 10 mM dTTP, dCTP, dGTP, dATP Ethanol 100 (20C) Ethanol 70, 95, 100 Fluorescein-11-dATP 2.5% glutaraldehyde Igepal CA-630 LR-White 6 mM; 50 mM MgCl2 9 NaCl 4% paraformaldehyde PBS PBS-glycerol 0.1 M phosphate buffer 10X PCR buffer Proteinase K 10 M anti-sense primer 10 M sense primer Anti-sense probe Sense probe 10 mg/ml tRNA 40 U/l RNasin 5X RT buffer 20X SSC Streptavidin conjugated to colloidal gold 5 U/l Taq DNA polymerase TrisHCl buffer TrisHCl/CaCl2 buffer TrisHCl/NaCl buffer Tris-HCl/NaCl/MgCl2 buffer DEPC water Sterile water 200 U/l reverse transcriptase 10.6.2.2 Fixation
See Appendix B2.3. See Appendix B2.5. See Appendix B2.7. dNTP
See Appendix B4.2. See Appendix B5.3.2. See Appendix B2.12. See Appendix B2.19. See Appendix B4.3.2. See Appendix B3.4.3. See Appendix B8.1.1. See Appendix B3.4.1. See Appendix B2.14.1. See Section 5.3.2. See Section 5.3.2. See Section 6.3. See Section 6.3. See Appendix B2.15.
See Appendix B3.5.
See Appendix B3.7.1. See Appendix B3.7.2. See Appendix B3.7.5. See Appendix B3.7.4. See Appendix B1.2. See Appendix B1.1.
a. Fix the sample in 4% 2h paraformaldehyde in phosphate buffer, using the conditions mentioned above. b. Rinse in phosphate buffer. 2 5 min 10.6.2.3 Sections
According to the size of the sample Addition of a low percentage of glutaraldehyde (e.g., 0.1%) (see Section 2.1.3.3)
a. Adhere the sample to the object holder of a vibratome.
Use cyanolit-type glue.
317
Typical Protocols b. Immerse the sample in 2X SSC buffer to obtain 50- to 70-m-thick oating sections. c. Place two to ve sections in 2X SSC buffer in microtubes. The area of the section must be large enough not to hamper subsequent procedures. All the different RT, PCR, and hybridization steps are carried out on oating sections in microtubes.
10.6.2.4
Pretreatments 5 min 37C 5 g/ml 15 min 37C rt
a. Incubate the sections in TrisHCl/NaCl/CaCl2 buffer. b. Add proteinase K.
c. Rinse in Tris-HCl/NaCl/CaCl2 buffer. d. Transfer the sections to other microtubes containing 0.1 M phosphate buffer, pH 7.4. 10.6.2.5 Reverse transcription
Reaction mixture a. In a sterile microtube, prepare the reaction mixture: 5X RT buffer 20 l 0.1 M DTT 10 l 10 mM dNTPs 5 l 40 U/l RNasin 2.5 l 10 M anti-sense primer 10 l Sterile water 47.5 l b. Replace the phosphate buffer with the reaction mixture, and pipette carefully to suspend the sections. c. Add 200 U/l reverse 5 l transcriptase. d. Incubate in a thermocycler. 1h 37C 1h 42C e. Deactivate the enzyme. 2 min 94C Washing The sections are rinsed in 5 min phosphate buffer. 10.6.2.6 Amplication
Final concentration: 1X Final concentration: 10 mM Final concentration: 0.5 mM Final concentration: 1 U/l Final concentration: 1 M To a nal volume of 95 l
Final concentration: 10 U/l Final volume: 100 l If MMLV is the reverse transcriptase used If AMV is the reverse transcriptase used The temperature at which the enzyme is destroyed
Reaction mixture a. In a sterile microtube placed in ice, prepare the reaction mixture: 10X PCR buffer 10 l 25 mM MgCl2 6 l 10 mM dNTP mixture 5 l 318
Final concentration: 1X Final concentration: 1.5 mM Final concentration: 0.2 mM
10.6 10 M labeled sense primer
Indirect in Situ RT-PCR Using Electron Microscopy Final concentration: 1 M Possible to use only one labeled primer, but to this extent the effectiveness of the amplication will decline Final concentration: 1 M To a nal volume of 96 l
10 l
10 M labeled anti-sense primer 10 l Sterile water 55 l b. Replace the phosphate buffer with the reaction mixture, and suspend the sections by careful pipetting. The hot start
The specicity of primer matching must be ensured. There is a high risk of nonspecic hybridization at low temperature. Final concentration: 0.2 U/l Final volume: 100 l To facilitate the subsequent washing steps, it is best not to add mineral oil. The number of cycles must be optimized for each operation.
a. Incubate b. Add 5 U/l Taq DNA polymerase, and check that the tube is closed. Amplication cycles a. Program 20 amplication cycles: Denaturing Hybridization
5 min 82C 4 l
1 min 94C 90 s 50C 90 s 72C 5 min 72C 10 s 30C
The hybridization temperature must be optimized in keeping with the characteristics of the primers being used.
Extension b. Perform nal extension. c. Stop the reaction.
The sections can be stored at 4C in a thermocycler.
Washing a. Remove all the reaction medium. b. Suspend the sections in 2 10 min phosphate buffer. 10.6.2.7 Hybridization
The amplied products are detected by hybridization, using two specic oligonucleotidic probes labeled with digoxygenin. Reaction mixture a. In a sterile microtube placed in ice, prepare the reaction mixture: 20X SSC 100 l Deionized formamide 250 l 50X Denhardts solution 10 l 10 mg/ml tRNA 12.5 l
See Chapter 6. Fluorescein or biotin
Final concentration: 4X Final concentration: 50% Final concentration: 1X Final concentration: 250 g/ml
319
Typical Protocols 10 mg/ml salmon sperm DNA 12.5 l Labeled sense probe 8 l (1.25 pmol/l) Labeled anti-sense probe 8 l (1.25 pmol/l) Sterile water 99 l b. Replace the phosphate buffer with the reaction mixture. c. Suspend the sections. d. Denature. 5 min 95C e. Cool immediately on ice. 5 min f. Incubate. Overnight 40C Final concentration: 250 g/ml Final concentration: 20 pmol/ml Final concentration: 20 pmol/ml To a nal volume of 500 l
On a heating block To stabilize the DNA in single-strand form In the thermocycler Necessary that the hybridization temperature take into account the characteristics of the probes See Chapter 6, Figure 6.4. It is always possible to increase or decrease the concentration of the SSC buffer and the washing time in line with the results obtained.
Washing 2X SSC 1X SSC 0.5X SSC Postxation 4% paraformaldehyde in 2X SSC buffer Rinsing in 2X SSC 10.6.2.8 Embedding
30 min rt 30 min rt 30 min rt 10 min 3 5 min
To x the hybrids that have been formed, and to stabilize the tissue and cell structures
Vibratome sections are embedded in LR-White resin, or any other hydrophilic resin. Dehydration Alcohol 50, 70, 95, 100 10 min per bath Substitution Alcohol 100LR White 30 min (2:1 v/v) Alcohol 100LR White 30 min (1:1 v/v) Alcohol 100LR White 30 min (1:2 v/v) Polymerization The sections, spread on a at 2 days surface in a drop of resin, 60C are covered with a capsule.
Of the Unicryl or Lowicryl type
Polymerization can take place only in anaerobic conditions. See Section 8.11.
320
10.6 10.6.2.9 Ultramicrotomy
In this procedure, 100-nm sections are made on an ultracut equipped with a diamond knife, and placed on collodionized, carbonated nickel grids. 10.6.2.10 Immunocytological detection
See Section 8.11.4.
Indirect detection by an antibody directed against the antigenic molecule used a. Incubate: Mouse anti-label monoclonal 1h antibody at a dilution of 1:50 in rt TrisHCl/NaCl buffer added to a blocking agent and a detergent b. Rinse: In the same buffer, but without detergent In TrisHCl/NaCl buffer, pH 8.2, added to a blocking agent and a detergent 2 5 min 2 5 min
According to the label The grids are incubated on drops of reagent, with the section placed on the liquid. Digoxygenin, uorescein, or biotin Example: 0.5% ovalbumin Example: 0.05% Tween 20
c. Incubate: Anti-mouse antibody conjugated 1h to 10 nm colloidal gold at a rt dilution of 1:50 in the same buffer d. Rinse: In TrisHCl/NaCl buffer, 2 5 min pH 8.2 In 2X SSC buffer 2 5 min Fixation 2.5% glutaraldehyde in 2X SSC 5 min buffer Washing in 2X SSC 2 5 min Rapid rinsing in distilled water Contrast By 5% aqueous uranyl acetate 30 min Rinsing in distilled water Drying the grids 10.6.2.11 Observations
A pH of 8.2 is important for the stability of an antibody conjugated to colloidal gold. Example: 0.5% ovalbumin Example: 0.05% Tween 20 Depending on the primary antibody used The intensity of the labeling inversely proportional to the size of the gold particles
. Nonsterile, but ltered on 0.2 m Nonsterile, but ltered on 0.2 m
The grids are observed by transmission electron microscopy. 10.6.2.12 Controls
The observation voltage must be low, i.e., 60 to 80 kV.
Positive controls Tissue containing the mRNA being sought
The only way to nd out if the reaction has taken place normally in the case of negative results 321
Typical Protocols Negative controls Tissue that is known not to contain the mRNA being sought Reaction controls Omission of reverse transcriptase, or treatment of tissue with RNase Omission of Taq DNA polymerase Omission of primers
To conrm a specic amplication Control of the reverse transcription If the amplication step omitted, no positive result Possible that a weak signal may, however, appear, due to the endogenous reparative power of Taq DNA polymerase Detection control If the amplied products have diffused, a band will be clearly visible on the gel.
Omission of the primary antibody Diffusion control 10 l of the reaction medium is removed after amplication, and subjected to electrophoresis.
322
Chapter 11
Examples of Observations
FIGURE 11.1 (Color Figure 11.1 follows page 336.)

Title Methods A comparison between indirect in situ hybridization and direct and indirect in situ PCR for the detection of human papilloma virus (HPV) DNA A: In situ hybridization B: Direct in situ PCR C: Indirect in situ RT-PCR Squamous intraepithelial lesion (human condyloma) 4% paraformaldehyde Adjacent 4-m sections 5 g/ml
Tissue Fixation Tissue preparation Parafn embedding Sections Pretreatment Proteinase K Amplication A: Control by in situ hybridization B: Direct in situ PCR
C: Indirect in situ PCR Detection In situ hybridization and/or Immunohistochemistry Bar Comments
Fluorescent cDNA probe 20-mer sense and anti-sense primers Incorporation of dUTP-11-biotin 25 cycles 20-mer sense and anti-sense primers 25 cycles Examples of Observation Alkaline phosphatase conjugated anti-uorescein Streptavidin/alkaline phosphatase/NBT-BCIP 10 m The amplied product (dark purple precipitate) is detected on three adjacent sections in the cells of the cervical epithelium, which have perinuclear halos and atypical nuclei. Furthermore, no viral DNA is visible in the basal cells after in situ hybridization alone (A). All the cells are strongly positive after direct in situ PCR (B). The signal is more specic after indirect in situ PCR (C).
325
326 Examples of Observations Title Method Monolayer culture Fixation Pretreatment Proteinase K Amplication A: Direct in situ PCR 1 g/ml Biotinylated dATP 20-mer sense and anti-sense primers Incorporation of dUTP-11-biotin 25 cycles 20-mer sense and anti-sense primers 25 cycles Detection of HPV 6 DNA in HeLa cells culture A: Direct in situ PCR HeLa cells 4% paraformaldehyde B: Indirect in situ PCR C: Control without Taq ADN polymerase Detection Immunohistochemistry Bar Comments Streptavidin/alkaline phosphatase/NBT-BCIP 10 m HeLa cells contain, on average, more than ten copies of the virus per genome. The direct (A) and the indirect (B) method give a strong signal in all the cells. No signal is found when Taq polymerase is omitted (C).

Title Method Monolayer culture Fixation Pretreatment Proteinase K Amplication Indirect in situ PCR 1 g/ml 20-mer sense and anti-sense primers 25 cycles Biotinylated HPV 6 cDNA probe Streptavidin/alkaline phosphatase/NBT-BCIP 10 m Only the HeLa cells, which are characterized by their broblastic shapes, have positive nuclei (see Figure 11.2). Those of the Hep2 cells, which are characterized by their epidermoid appearance, remain negative after amplication. This internal control demonstrates the specicity of the detection method. Examples of Observation Detection In situ hybridization, and Immunohistochemistry Bar Comments The detection of HPV 6 DNA in a coculture of two different cell types Indirect in situ PCR HeLa and Hep2 cells 4% paraformaldehyde
327
328 Examples of Observations Title A demonstration of the specicity of the detection of the mRNA that codes for the growth hormone receptor in the pituitary gland Indirect in situ RT-PCR Rat pituitary 7 m 4% paraformaldehyde 1 g/ml 30-mer anti-sense probe 25-mer anti-sense probe 25-mer sense and anti-sense probes 25 cycles Treatment by RNase 30-mer sense and anti-sense oligonucleotide probes Antibody/alkaline phosphatase/NBT-BCIP 10 m After the indirect RT-PCR reaction (A), the signal is very strong and it is important to note that there are still some negative cells. A little more than half the cells in the anterior lobe of the pituitary are detected after the RT reaction (B) and after in situ hybridization (C). After the destruction of the RNA before the RTPCR, no signal is detectable (D). Method Tissue Tissue preparation Frozen sections Fixation Pretreatment Proteinase K Amplication A: Indirect in situ RT-PCR B: Reverse transcription C: In situ hybridization D: Control Detection In situ hybridization, and Immunohistochemistry Bar Comments

Title The effect of the number of reaction cycles on the amplication of the mRNA that codes for the growth hormone receptor in the pituitary Indirect in situ RT-PCR Rat pituitary 5 m 4% paraformaldehyde 3 g/ml 25-mer sense and anti-sense primers
Method Tissue Tissue preparation Parafn-embedded sections Fixation Pretreatment Proteinase K Amplication A: 5 cycles B: 15 cycles C: 25 cycles Detection In situ hybridization, and Immunohistochemistry Bar Comments
30-mer sense and anti-sense probes labeled with digoxigenin Anti-digoxigenin/alkaline phosphatase/NBT-BCIP 10 m The number of cycles has no effect on the percentage of positive cells observed (see Figure 11.4), but a higher number of cycles produces a stronger signal: A (5 cycles) < B (15 cycles) < C (25 cycles).
Examples of Observation
329
330 Examples of Observations Title The detection of weakly expressed mRNA coding for extrapituitary growth hormone (GH) in a lymphoid organ, namely, the spleen Indirect in situ RT-PCR Rat spleen 5 m 4% paraformaldehyde 3 g/ml 25-mer sense and anti-sense primers Omission of the RT step and Taq DNA polymerase Method Tissue Tissue preparation Parafn-embedded sections Fixation Pretreatment Proteinase K Amplication A: Indirect in situ RT-PCR B: Control without amplication Detection In situ hybridization Immunohistochemistry Bar Comments 30-mer sense and anti-sense probes labeled with digoxigenin Anti-digoxigenin/alkaline phosphatase/NBT-BCIP 10 m GH mRNA synthesis occurs in the lymphoid white pulp that surrounds and follows the arteries, and in the lymphocytes of the red pulp. The reticular cells of the connective network remain negative (A). No signal is detected in the control (B). (From Recher, S. et al., J. Histochem. Cytochem., 49, 347, 2001. With permission.)

Title Method Tissue 5 m 4% paraformaldehyde 3 g/ml 25-mer sense and anti-sense primers 25 cycles Tissue preparation Parafn-embedded sections Fixation Pretreatment Proteinase K Amplication Indirect in situ RT-PCR Detection In situ hybridization, and Immunohistochemistry Bar Comments GH gene expression in adult rat thymus and Peyers patches Indirect in situ RT-PCR A: Adult rat thymus B: A Peyers patch in an adult rat
30-mer sense and anti-sense probes labeled with digoxigenin Anti-digoxigenin/alkaline phosphatase/NBT-BCIP 10 m GH-gene-expressing cells are mainly found in the thymic cortex, while the reticular and epithelial cells of the medulla are negative, and act as an internal control (A). In Peyers patches, the signal is specically associated with all the lymphocytes, whereas epithelial cells of the terminal ileum remained negative (B). (From Recher, S. et al., J. Histochem. Cytochem., 49, 347, 2001. With permission.)
331
332 Examples of Observations Title Detection of mRNA coding for the growth hormone in the rat fetal thymus Indirect in situ RT-PCR 18-day-old rat fetal thymus 5 m 4% paraformaldehyde 3 g/ml 25-mer anti-sense primer 25-mer sense and anti-sense primers Omission of Taq DNA polymerase Method Tissue Tissue preparation Parafn-embedded sections Fixation Pretreatment Proteinase K Amplication A: Indirect in situ RT-PCR B: Control without amplication Detection In situ hybridization, and Immunohistochemistry Bar Comments 30-mer sense and anti-sense olidonucleotide probes labeled with digoxigenin Anti-digoxigenin/alkaline phosphatase/NBT-BCIP 10 m GH gene expression is found in all the fetal thymus cells. However, the lymphocytes located in the cortical region of the thymus lobe give a stronger signal (A). In situ RT-PCR performed without Taq DNA polymerase on an adjacent section is used as a negative control (B). (From Recher, S. et al., J. Histochem. Cytochem., 49, 347, 2001. With permission.)

Title The detection of mRNA coding for the growth hormone (GH) in rat fetal liver Indirect in situ RT-PCR 18-day-old rat fetal liver 5 m 4% paraformaldehyde 3 g/ml 25-mer anti-sense primer 25-mer sense and anti-sense primers
Method Tissue Tissue preparation Parafn-embedded sections Fixation Pretreatment Proteinase K Amplication RT Indirect in situ PCR Detection In situ hybridization, and Immunohistochemistry Bar Comments
30-mer sense and anti-sense oligonucleotide probes labeled with digoxigenin Anti-digoxigenin/alkaline phosphatase/NBT-BCIP 10 m In the fetus, the liver has a hematopoietic function. Here, a strong signal demonstrating GH gene expression is detected in the hematopoietic cells around the centrolobular vein, whereas the hepatocytes remain consistently negative. (From Recher, S. et al., J. Histochem. Cytochem., 49, 347, 2001. With permission.)
333
334 Examples of Observations Title The location of mRNA coding for extrapituitary growth hormone in a benign mammary pathology, namely, broadenoma Indirect in situ RT-PCR Fibroadenoma in a human mammary gland 5 m 4% formol 3 g/ml 25-mer anti-sense primer 25-mer sense and anti-sense primers Control without amplication Method Tissue Tissue preparation Parafn-embedded sections Fixation Pretreatment Proteinase K Amplication A: RT Indirect in situ PCR B: Omission of Taq DNA polymerase Detection In situ hybridization, and Immunohistochemistry Bar Comments 30-mer sense and anti-sense oligonucleotide probes labeled with digoxigenin Anti-digoxigenin/alkaline phosphatase/NBT-BCIP 10 m All the proliferative epithelial cells and broblasts are positive (A). The specicity of the reaction is demonstrated by the absence of a signal when Taq DNA polymerase is omitted (B).

Title The detection of mRNA coding for leghemoglobin in a secondary root Indirect in situ RT-PCR Roots of Medicago truncatula
70 m 4% paraformaldehyde 2 g/ml 25-mer anti-sense primer 25-mer sense and anti-sense primers
Method Tissue A: Secondary root B: Degenerative nodule Tissue preparation Vibratome sections Fixation Pretreatment Proteinase K Amplication RT Indirect in situ PCR Detection In situ hybridization, and Immunohistochemistry Bar Comments
cDNA sense and anti-sense probes labeled with digoxigenin Anti-digoxigenin/alkaline phosphatase/NBT-BCIP 10 m Only the mesenchymatous cells in the secondary root give a positive signal (A), whereas the cells in a degenerative nodule are negative (B). (From De Billy, F., unpublished data. With permission.)
335
336 Title Method Examples of Observations Detection, on semithin sections, of pituitary mRNA coding for the growth hormone receptor Indirect in situ RT-PCR in electron microscopy: Pre-embedding method Frontal lobe of rat pituitary 4% paraformaldehyde 50 m 1 g/ml 0.01% 25-mer anti-sense primer 25-mer sense and anti-sense primers 20 cycles Control without amplication Tissue Fixation Tissue preparation Vibratome sections Pretreatments Proteinase K Triton X100 Amplication A: RT Indirect in situ PCR B: Omission of Taq DNA polymerase Detection In situ hybridization, and Immunocytochemistry Bar Comments Before embedding 30-mer sense and anti-sense probes labeled with digoxigenin Antibody/peroxidase/DAB 10 m Only a few cells are positive in this semithin transversal section of the thick section of the anterior lobe of the pituitary (insert). No signal (dense product) is visible in this section other than in the cytoplasm of certain cells (A). In the absence of amplication, there are no positive cells (B).
FIGURE 11.12
FIGURE 11.13
Title Method Ultrastructural detection of mRNA coding for the growth hormone receptor in the pituitary Indirect in situ RT-PCR in electron microscopy. Hydrophilic resin postembedding method Rat pituitary 4% paraformaldehyde LR White 1 g/ml
Tissue Fixation Tissue preparation Hydrophilic resin embedding Pretreatment Proteinase K Amplication A: In situ hybridization B: RT In situ PCR In situ hybridization Detection Immunocytology Bar Comments
30-mer sense and anti-sense probes labeled with digoxigenin 25-mer anti-sense primer 25-mer sense and anti-sense primers 20 cycles 30-mer sense and anti-sense probes labeled with digoxigenin Antibody/colloidal gold (10 nm) 1 m The colloidal gold particles (arrow) are present in the cytoplasmic matrix, near the endoplasmic reticulum (er) of three cell types: gonadotrophs (LH-FSH), lactotrophs (PRL), and somatotrophs (GH). A comparison between the labeling obtained without amplication, after in situ hybridization (A) and after amplication (B), shows no signicant increase in the density of the colloidal gold particles.
337
338 Title Method Examples of Observations Ultrastructural detection of mRNA coding for the growth hormone receptor in the pituitary Indirect in situ RT-PCR in electron microscopy: Non-embedding method Rat pituitary 4% paraformaldehyde Ultrathin frozen tissue sections (80 nm) None Tissue Fixation Tissue preparation Freezing Cryoultramicrotomy Pretreatment Amplication A: In situ hybridization B: RT In situ PCR In situ hybridization Detection Immunocytology Bar Comments 30-mer sense and anti-sense probes labeled with digoxigenin 25-mer anti-sense primer 25-mer sense and anti-sense primers Limited to ve cycles 30-mer sense and anti-sense probes labeled with digoxigenin Antibody/colloidal gold (10 nm) 1 m The colloidal gold particles (arrows) are present in the same cells, close to the endoplasmic reticulum (er), both without amplication, after in situ hybridization alone (A), and after amplication (B). No signicant increase in the density of colloidal gold particles is observed with this non-embedding amplication method.
FIGURE 11.14
FIGURE 11.15
Title Method Ultrastructural detection of mRNA coding for the growth hormone receptor in the pituitary Indirect in situ RT-PCR in electron microscopy: Preembedding method Rat pituitary 4% paraformaldehyde 50 m 1 g/ml 0.01% 25-mer anti-sense primer 25-mer sense and anti-sense primers 20 cycles
Tissue Fixation Tissue preparation Vibratome sections Pretreatments Proteinase K Triton X100 Amplication RT In situ PCR Detection In situ hybridization Immunocytology Bar Comments
30-mer sense and anti-sense probes labeled with digoxigenin After embedding in hydrophilic resin (LR White) Antibody/colloidal gold (10 nm) 1 m The colloidal gold particles are present in the cytoplasmic matrix, close to the endoplasmic reticulum (er), in the gonadotrophs (LH-FSH), lactotrophs (PRL), and somatotrophs (GH). The density of the labeling is higher with this preembedding amplication method.
339
340 Title Method Examples of Observations Cellular specicity of the ultrastructural detection of the mRNA coding for the growth hormone receptor in the pituitary Indirect in situ RT-PCR in electron microscopy: Preembedding method Rat pituitary 4% paraformaldehyde 50 m 1 g/ml 0.01% 25-mer anti-sense primer 25-mer sense and anti-sense primers 20 cycles Tissue Fixation Tissue preparation Vibratome sections Pretreatments Proteinase K Triton X100 Amplication RT In situ PCR Detection In situ hybridization Immunocytology Bar Comments 30-mer sense and anti-sense probes labeled with digoxigenin After embedding in hydrophilic resin Antibody/colloidal gold (10 nm) 1 m Colloidal gold particles are present in the cytoplasm of a somatotroph (GH), but not in any corticotrophs (ACTH) or thyrotrophs (insert), which means that the amplied products have not diffused between different types of cell.
FIGURE 11.16
FIGURE 11.17
Title Method The double ultrastructural detection of mRNA coding for the growth hormone and prolactin in lactotrophs Indirect in situ RT-PCR in electron microscopy: Preembedding method Immunocytology after embedding Rat pituitary 4% paraformaldehyde 50 m 1 g/ml 0.01% 25-mer anti-sense primer 25-mer sense and anti-sense primers
Tissue Fixation Tissue preparation Vibratome sections Pretreatments Proteinase K Triton X100 Amplication RT In situ PCR Detection RT-PCR In situ hybridization, and Immunocytology Immunocytology Bar Comments
30-mer sense and anti-sense probes labeled with digoxigenin After embedding Antibody/colloidal gold (10 nm) Anti-prolactin (PRL) serum Colloidal gold (5 nm) 1 m 10-nm colloidal gold particles that are used to visualize mRNA coding for the growth hormone are present in the cytoplasm and the nucleus (N) of a lactotroph (PRL), which is identied by the immunocytological detection of the content of its secretion grains (5-nm gold particles).
341
Appendices
CONTENTS
A EQUIPMENT
A1 Practical Precautions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A1.1 RNase-Free Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A1.2 Chemical Risks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A1.3 Radioactive Risks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A2 Sterilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A3 Pretreatment of Slides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A4 Electrophoresis of PCR Products in Agarose Gel. . . . . . . . . . . . . . . . 349 349 350 351 351 352 353
B REAGENTS
B1 Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B1.1 Sterile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B1.2 Diethylpyrocarbonate (DEPC) . . . . . . . . . . . . . . . . . . . . . . . . B2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.1 Agarose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.2 Ammonium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.3 Calcium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.3.1 Calcium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . B2.3.2 Calcium Chloride/Cobalt Chloride . . . . . . . . . . . . . B2.4 Deionized Formamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.5 Denhardts Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.6 Dextran Sulfate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.7 Dithiothreitol (DTT). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.8 DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.9 DNase I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.10 Ethylene Diamine Tetra-Acetic Acid (EDTA) . . . . . . . . . . . . B2.11 Lithium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.12 Magnesium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.13 Poly (A) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.14 Proteinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.14.1 Proteinase K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.14.2 Pepsine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.14.3 Pronase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.15 RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.16 RNase A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.17 Sarcosyl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.18 Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355 355 355 355 355 356 356 356 357 357 357 358 358 359 359 359 360 360 360 360 360 361 361 361 362 362 362 345
Appendices B2.19 Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.20 Sodium Hydroxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.21 Tris . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.22 Triton X-100. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Buffers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.1 Acetylation Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.2 Cacodylate Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.3 DNase I Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.4 Phosphate Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.4.1 1 M Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.4.2 Phosphate/NaCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.4.3 PBS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.5 SSC Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.6 TE (TrisEDTA) Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.6.1 TE Buffer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.6.2 TENaCl Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.7 TrisHCl Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.7.1 TrisHCl Buffer. . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.7.2 TrisHCl/CaCl2 Buffer . . . . . . . . . . . . . . . . . . . . . . B3.7.3 TrisHCl/Glycine Buffer. . . . . . . . . . . . . . . . . . . . . B3.7.4 TrisHCl/MgCl2 Buffer . . . . . . . . . . . . . . . . . . . . . . B3.7.5 TrisHCl/NaCl Buffer . . . . . . . . . . . . . . . . . . . . . . . B3.7.6 TrisHCl/NaCl/MgCl2 Buffer . . . . . . . . . . . . . . . . . Fixatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B4.1 Formol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B4.1.1 Neutral Buffered Formalin . . . . . . . . . . . . . . . . . . . B4.1.2 Formol Saline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B4.2 Glutaraldehyde (2.5%) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B4.3 Paraformaldehyde. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B4.3.1 Paraformaldehyde 40% . . . . . . . . . . . . . . . . . . . . . . B4.3.2 Paraformaldehyde 4% . . . . . . . . . . . . . . . . . . . . . . . B4.3.3 Paraformaldehyde 4%/Glutaraldehyde 0.05% . . . . Embedding Media. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B5.1 Materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B5.2 Epoxy Resins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B5.2.1 Eponaraldite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B5.2.2 Epon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B5.3 Acrylic Resins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B5.3.1 Lowicryl K4M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . B5.3.2 LR White Medium. . . . . . . . . . . . . . . . . . . . . . . . . . B5.3.3 Unicryl. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Revelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B6.1 Autoradiography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B6.1.1 Standard Developer . . . . . . . . . . . . . . . . . . . . . . . . . B6.1.2 Fixative . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B6.2 Immunocytology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B6.2.1 Blocking Solutions . . . . . . . . . . . . . . . . . . . . . . . . . B6.2.1.1 Nonspecic Sites . . . . . . . . . . . . . . . . . . B6.2.1.2 Endogenous Alkaline Phosphatases . . . B6.2.1.3 Endogenous Peroxidases . . . . . . . . . . . . 363 363 363 364 364 364 364 365 365 365 366 366 367 367 367 368 368 368 369 369 369 370 370 371 371 371 371 371 372 372 372 373 373 373 374 374 374 375 375 376 376 377 377 377 377 378 378 378 378 379
B3
B4
B5
B6
346
Appendices Chromogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B6.2.2.1 Alkaline Phosphatase . . . . . . . . . . . . . . B6.2.2.2 Peroxidase . . . . . . . . . . . . . . . . . . . . . . . B7 Stains/Coating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B7.1 Light Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B7.1.1 Cresyl Violet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B7.1.2 Eosin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B7.1.3 Harriss Hematoxylin . . . . . . . . . . . . . . . . . . . . . . . B7.1.4 Methylene Green . . . . . . . . . . . . . . . . . . . . . . . . . . . B7.1.5 Rapid Nuclear Red . . . . . . . . . . . . . . . . . . . . . . . . . B7.1.6 Toluidine Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B7.2 Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B7.2.1 Uranyl Acetate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . B7.2.1.1 2.5% Alcoholic Uranyl Acetate . . . . . . . B7.2.1.2 2 to 5% Aqueous Uranyl Acetate . . . . . B7.2.1.3 4% Neutral Uranyl Acetate . . . . . . . . . . B7.2.2 Lead Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B7.2.3 Methylcellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . B7.2.3.1 2% Methylcellulose. . . . . . . . . . . . . . . . B7.2.3.2 0.8% Methylcellulose, 0.2% Neutral Uranyl Acetate . . . . . . . . . . . . . B7.2.4 Sodium Silicotungstate (0.5%) . . . . . . . . . . . . . . . . B8 Mounting Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B8.1 Aqueous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B8.1.1 Buffered Glycerine . . . . . . . . . . . . . . . . . . . . . . . . . B8.1.2 Moviol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B8.2 Permanent. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B6.2.2 379 379 380 380 380 380 381 381 382 382 382 383 383 383 383 383 384 384 384 385 385 386 386 386 386 387
347
Appendix A
EQUIPMENT
This appendix presents the procedures for preparing materials for in situ PCR/RT-PCR to obtain the best preservation of nucleic acids.
A1 PRACTICAL PRECAUTIONS
A1.1 RNase-Free Conditions
Reagents are available that destroy RNases. RNase activity is inhibited in the buffer (presence of RNasin ). Commercial solutions for inhibiting RNases are available. This buffer, which is very stable, inhibits the action of enzymes that break down DNA. Instruments must be sterile. Slides can be dried at room temperature. Storage in the absence of water is the best way of inhibiting RNases. RNases and DNases act only in the presence of water. Place the dry slides in a airtight box containing desiccant (silicagel). Prefer the storage in parafn block. Place the dry slides in an airtight box containing a desiccant (silicagel) at room temperature or 20C. Do not open the box until it is at room temperature, to limit condensation on the sections. At 4C under a drop of sucrose used to pick up the sections Limited storage: 1 to 2 weeks Do not store these sections for more than 1 week.
Working areas Areas close to sterility Probe Use of all the probes
Storage DEPC-treated water Choice of TE buffer (TE/NaCl) Tissue Sampling in sterile conditions Storage of sections
Sections of tissue embedded in parafn
Frozen sections
Semithin frozen sections
Ultrathin sections
349
Equipment Equipment/Reagents/Solutions Eppendorf tubes Sterilized cones Gloves New reagents Recipients
Water treated with DEPC or containing RNase inhibitors Conclusion Avoiding RNase contamination is easier than removing it.
Disposable is preferable to sterilized equipment. Do not touch the equipment or the reagents without gloves. Reserved exclusively for in situ PCR/RTPCR. Reserved for in situ PCR/RT-PCR and sterilized immediately after use (otherwise, disposable equipment). The risk of contamination increases with time, in relation to the frequency of opening.
A1.2 Chemical Risks

Reagents/Resins Reagents For electron microscopy a number of products are used that are dangerous if touched or inhaled: Formaldehyde Glutaraldehyde Some organic solvents must be used in a ventilated area that vents to the outside. Other products, such as: Formamide Lead nitrate Sodium cacodylate Uranyl acetate Toxic vapors (isoamyl acetate, xylene, acetone, etc.) In accordance with the manufacturers instructions All experiments must be carried out under a fume hood.
radiation Any trace of these products on the skin must be carefully washed off in running water. Wear a mask. Avoid all contact with the skin. In the case of spills, wash hands thoroughly with soapy water. Never mouth-pipette these products.
are highly toxic if inhaled or ingested. They must be used with great care. Resins Epoxy and acrylic resins are carcinogenic and highly toxic. Skin contact is dangerous.
Storage/Waste products Glassware, waste chemicals, and biological material for incineration are collected in special waste containers.
350
Appendix A
A1.3 Radioactive Risks

Radioactive hazards are of different origins: Gloves Protection/control Storage of radioactive sources Radioprotection training courses
Contact the person responsible. Change regularly to prevent contamination. Gloves are not a barrier to radiation. Regularly check surfaces, screens, hands 35 33 and equipment for contamination ( S, P). Store in containers stored in a special room. It is necessary to be familiar with safety precautions.
Table A1
Summary Table of Precautions to Be Taken during the Use of Radioisotopes

Special equipment Screens: 0.2 mm glass, or 0.3 mm Plexiglas Two pairs of gloves to be worn Contamination Risks, controls No irradiation
Isotopes
35 33
Wear a lm badge No
S P
Precautions Soak contaminated material in a diluted solution of decontaminant, then wash in running water Control/Wastes After radioactive decay (>10 half-lives) waste can be disposed of as nonradioactive waste.
Decontamination Disposal must follow Health and Safety protocols.
A2 STERILIZATION
The sterilization of solutions and of small equipment is indispensable for in situ PCR/RT-PCR. Equipment Aluminum foil Autoclave Autoclave tape Oven Minor equipment Glass staining trays Maintenance of clean conditions and the use of solutions exclusively for in situ PCR/RT-PCR
The surface pattern changes after sterilization. Glass equipment is autoclaved or sterilized in an oven at 50C.
351
Equipment Protocol Treatment of equipment, including magnetic bars, in: Oven Autoclave 2h 180C 2h 105C or 30 min 125C RNases are almost completely destroyed by this treatment. Two bars. Allow the equipment to cool in the oven (risk of breakage if it is placed on a cold surface).
A3 PRETREATMENT OF SLIDES
Equipment Metal forceps Oven or autoclave Trays, slide holders Slides Reagents Acetone Alcohol 95% 3-Amino-propyl-tri-ethoxy-silane Hydrochloric acid 10 N Sterile distilled water Solutions Cleaning: Alcohol/HCl (5 ml HCl for 1 l 95% alcohol) Treatment: 3-Amino-propyl-tri-ethoxy-silane at 2% in acetone Precaution Gloves should be worn throughout slide preparation. Protocol a. Wash: Alcohol/HCl Overnight Running water 1h Distilled water 1 min b. Dry the slides in oven. 180C 60 min c. Allow to cool. d. Immerse in treatment solution. 515 s e. Wash: Acetone 2 1 min Sterile water 1 min f. Dry. Overnight 42C Storage rt Up to a year 352
See Appendix B1.1. Stable solution, which can be stored Solution unstable; prepare just before use.
Sterilization is necessary.
Room temperature Dust-free conditions
Appendix A
A4
ELECTROPHORESIS OF PCR PRODUCTS IN AGAROSE GEL
Equipment Electrophoresis apparatus Gel former tray Combs Tank Reagents TAE buffer Dissolve 242 g Tris base and 57.1 ml of glacial acetic acid in a nal volume of 900 ml of water Add 100 ml of 0.5 M EDTA; pH 8.0 Ethidium bromide Agarose for gel electrophoresis 6X DNA loading buffer: 30% (v/v) glycerol 0.25% (w/v) bromophenol blue in TE buffer pH 7.4 Ultraviolet transilluminator DNA size marker Protocol a. Weigh the appropriate amount of agarose and dissolve it in 100 ml of TAE buffer by boiling.
Stock solution: 50X
Stock solution: 10 mg/ml in water Or Orange G if the expected product is <300 bp Or TAE buffer Diluted to 50 ng/l in TE buffer (see Appendix B3.6) This can be done in a microwave oven. For PCR products of 300 to 1500 bp prepare 1.5% (w/v) agarose and for products <300 bp prepare 2.0% agarose. Wear protective gloves when handling ethidium bromide or gel containing it.
b. Allow the agarose to cool to about 60C and add 5 l of ethidium bromide stock solution for 100 ml of agarose. c. Allow any bubbles to disperse and pour the agarose into a gel former tray appropriate for the electrophoresis tank to be used, inserting combs to cast wells allowing for 20 to 25 l sample volume. d. Let the agarose solidify. 30 min rt e. Place the gel in the electrophoresis tank. f. Add sufcient TAE buffer to cover the electrodes and the gel. g. Mix 3 l of 6X loading buffer with 15 l of PCR product. h. Mix 3 l of 6X loading buffer with 15 l of size marker. i. Add the size marker to one lane of the gel and each sample to the other lanes.
Carefully pipette the sample into the well, below the surface of the TAE buffer. 353
Equipment j. Electrophorese the 3045 min samples until the dye 80100 V front has moved 3 to 4 cm 6070 mA down the gel. k. Examine the gel on an ultraviolet transilluminator and compare the PCR product band with the size marker to determine the size of the amplied product.
354
Appendix B
REAGENTS
B1 WATER
B1.1 Sterile
Sufcient for the detection of doublestranded DNA (e.g., viruses)
Reagents Distilled water Precaution Do not keep after opening. Protocol Sterilize in an autoclave. Storage
2h 105C At rt
Some weeks Indispensable C6H10O5
B1.2
Diethylpyrocarbonate (DEPC)
Reagents/Solutions DEPC Distilled water Precaution DEPC is dangerous. Protocol a. Mix: DEPC Water b. Shake under a fume hood. c. Sterilize in an autoclave. Storage
0.51 ml 1000 ml Overnight 30 min 105C At rt
Mw = 162.1
Some weeks
B2 SOLUTIONS
B2.1 Agarose
Solution stock: 2% Molecular-biology quality PBS buffer, phosphate, or cacodylate See Appendix B3.2 and B3.4.
Products/Solutions Agarose Fixation buffer Precaution Cover the container when the agarose is being dissolved to avoid evaporation.
355
Reagents Protocol a. Dissolve the agarose in the xation buffer: Agarose 2g Fixation buffer 100 ml b. After dissolving the agarose keep the solution in an oven. Storage Oven 56C
Use a water bath.
Keep at this temperature until use (some hours). Stock solution: 7.5 M CH3 CO2NH4 See Appendix B1.1.
B2.2
Ammonium Acetate
Reagents/Solutions Ammonium acetate 10 N hydrochloric acid Sterile water Precautions Check that the powder is dryvolatile reagent. Protocol Ammonium acetate 57.81 g Sterile water 80 ml a. Mix. b. Adjust the pH to 5.5 with HCl. Sterile water to 100 ml Sterilization Sterilize in an autoclave. 2h 105C Storage At 20C in aliquots of 50 to 100 l At room temperature for some months
Mw = 77.08
B2.3
B2.3.1
Calcium Chloride
Calcium chloride Stock solution: 1 M AR quality, to be used only for in situ PCR/ RT-PCR CaCl22H2O See Appendix B1.1.
Reagents/Solutions Calcium chloride Sterile water Precaution None Protocol Calcium chloride Sterile water Sterilization Sterilize in an autoclave. Storage
14.7 g to 100 ml 2h 105C At rt
Mw = 147.02
Some weeks before opening
356
Appendix B B2.3.2 Calcium chloride/cobalt chloride Reagents/Solutions Calcium chloride Cobalt chloride Sterile water Precaution None Protocol Calcium chloride 0.029 g Cobalt chloride 0.025 g Sterile water to 100 ml Sterilization Sterilize in an autoclave. 2h 105C Storage At rt Stock solution: 2 mM CaCl2 /2 mM CoCl2 CaCl22H2O CoCl2 See Appendix B1.1.
Mw = 147.02 Mw = 129.83
Some weeks before opening
B2.4
Deionized Formamide
Equipment Sterile ask Whatman lter N 1M Reagents/Solutions Amberlite resin, 20 to 50 mesh Formamide Precaution Avoid contact. Protocol Amberlite 5g Formamide lter 50 ml a. Shake gently. 30 min b. Filter on Whatman paper. Storage At 20C in sterile 1 ml tubes, and store in lightfree conditions.
Molecular-biology quality CH3NO Commercially available solution Mw = 45.04
Do not thaw. If it is liquid at 20C, it must not be used. Stock solution: 50X BSA, crystallized ve times Synthetic polymer of sucrose See Appendix B1.1.
B2.5
Denhardts Solution
Reagents/Solutions Bovine serum albumin (fraction V) Ficoll 400 Polyvinylpyrolidone Sterile water Precaution Risk of bacterial contamination Protocol a. Add: BSA Ficoll 400
1g 1g
357
Reagents Polyvinylpyrolidone 1g Sterile water to 100 ml b. Leave the mixture to hydrate overnight before shaking. c. Shake the mixture gently and intermittently for some days. Storage In aliquots. At 20C
Can be thawed and refrozen Stock solution: 50% Molecular-biology quality See Appendix B1.1.
B2.6
Dextran Sulfate
Reagents/Solutions Dextran sulfate Sterile water Precaution Reagent very difcult to pipette. Prepare extemporaneously only the quantity necessary. Protocol a. Add to a sterile Eppendorf tube: Dextran sulfate 50 mg Sterile water 65 l b. Mix. c. Microcentrifuge for a few seconds. Storage is possible In aliquots of 100 l At 20C
Mw = 500.00
It is recommended that it be stored in aliquots as the other components of the PCR/RT-PCR mixture should be. Stock solution: 1 M Molecular-biology quality C4H10O2S2 See Appendix B1.1. Denatured quickly in the PCR/RT-PCR buffer Mw = 154.24
B2.7
Dithiothreitol (DTT)
Reagents/Solutions DTT Sterile water Precaution Very volatile reagent, unstable at room temperature Does not resist denaturation Protocol a. Add to a sterile Eppendorf tube: Dithiothreitol 1.54 g Sterile water 10 ml b. Mix. Storage In aliquots of 100 l At 20C
Must never be refrozen. The strong smell indicates that the product is in good condition.
358
Appendix B
B2.8
DNA
Stock solution: 10 mg/ml sterile water Use commercially available preparations. See Appendix B1.1.
Reagents/Solutions Herring sperm DNA Salmon sperm DNA Sterile water Precaution Risk of bacterial contamination Protocol DNA Sterile water Sonicate for 10 min Storage
10 mg to 1 ml At 20C Maximum power May be thawed and refrozen Stock solution: 1 mg/ml Molecular-biology quality Check the activity of the enzyme. See Appendix B1.1.
B2.9
DNase I
Reagents/Solutions DNase I Sterile water Precaution Risk of RNase contamination Protocol Dissolve DNase I Sterile water Storage In aliquots of 100 l
1 mg 1 ml At 20C
To be expressed in units
B2.10
Ethylene Diamine Tetra-Acetic Acid (EDTA)
Stock solution: 500 mM RP quality, to be used only for in situ PCR/ RT-PCR C10H14N2O8Na22H2O or Titriplex III See Appendix B1.1.
Reagents/Solutions EDTA 10 N sodium hydroxide Sterile water Precaution Toxic reagent Protocol a. Dissolve EDTA 186 g Sterile water to 1 l b. Adjust the pH to 8.0 with sodium hydroxide. Sterilization Sterilize in an autoclave. 2h 105C Storage In aliquots At rt
Mw = 372.24
359
Reagents
B2.11
Lithium Chloride
Stock solution: 4 M RP quality, to be used only for in situ PCR/ RT-PCR LiCl See Appendix B1.1.
Reagents/Solutions Lithium chloride Sterile water Precaution Risk of bacterial contamination Protocol Lithium chloride Sterile water a. Mix b. Filter on a 0.22 m lter. Storage
8.48 g to 50 ml At 4C
Mw = 42.39
For a few weeks Stock solution: 1 M RP quality, to be used only for in situ PCR/ RT-PCR MgCl26H2O See Appendix B1.1.
B2.12
Magnesium Chloride
Reagents/Solutions Magnesium chloride Sterile water Precaution None Protocol Magnesium chloride Sterile water Sterilization Sterilize in an autoclave. Storage
20.3 g to 100 ml 2h 105C At rt
Mw = 203.30
B2.13
Poly (A)
Stock solution: 10 mg/ml See Appendix B1.2. C11H17N4O12 (polyribonucleotide adenylate) At 20C Some weeks
Reagents/Solutions DEPC-treated water Poly (A) Precaution Risk of hydrolysis by RNase Storage
B2.14
B2.14.1
Proteinase
Proteinase K Stock solution: 10 mg/ml
Reagents/Solutions Proteinase K Sterile water
See Appendix B1.1.
360
Appendix B Precaution Weak proteolysis at room temperature Protocol a. Place in a sterile Eppendorf tube: Proteinase K 10 mg Sterile water 1 ml b. Mix. Storage In aliquots of 100 l At 20C
Use commercially available solutions.
Dilute in TrisHCl/CaCl2 buffer (see Appendix B3.7.2). Stock solution: 10 mg/ml
B2.14.2 Pepsine Reagents/Solutions Pepsine Sterile water Precaution Weak proteolysis at room temperature Protocol a. Place in a sterile Eppendorf tube: Pepsine 10 mg Sterile water 1 ml b. Mix Storage In aliquots of 100 l At 20C
See Appendix B1.1. Use commercially available solutions.
Dilute in Tris/EDTA buffer 1X (see Appendix B3.6.1). Stock solution: 10 mg/ml
B2.14.3 Pronase Reagents/Solutions Pronase Sterile water Precaution Weak proteolysis at room temperature Protocol a. Place in a sterile Eppendorf tube: Pronase 10 mg Sterile water 1 ml b. Mix Storage In aliquots of 100 l At 20C
See Appendix B1.1. Use commercially available solutions.
Dilute in Tris/EDTA buffer 1X (see Appendix B3.6.1). Stock solution: 10 mg/ml sterile water See Appendix B1.2.
B2.15
RNA
Reagents/Solutions DEPC-treated water Yeast tRNA
361
Reagents Precaution Risk of hydrolysis Protocol RNA DEPC-treated water Sonicate 10 min. Storage
10 mg to 1 ml At 20C Maximum power May be thawed and refrozen Stock solution: 100 g/ml Molecular-biology quality Bovine pancreas ribonuclease See Appendix B1.1. See Appendix B3.6.
B2.16
RNase A
Reagents/Solutions RNase A Sterile water TE/NaCl buffer Precaution Risk of DNase contamination. Protocol RNase A Sterile water Storage In aliquots of 40 l
100 g 1 ml At 20C Solution stock: 20% buffered See Appendix B3.4. 0.2% Saponin may be used.
B2.17
Sarcosyl
Reagents Phosphate buffer or PBS Sarcosyl Precaution Toxic Protocol Sarcosyl Sterile water Storage
20 ml 80 ml At 4C
For some weeks Stock solution: 3 M AR quality, to be used only for in situ PCR/RT-PCR CH3 CO2H CH3 COONa See Appendix B1.1.
B2.18
Sodium Acetate
Reagents/Solutions Glacial acetic acid Sodium acetate Sterile water Precaution None Protocol Sodium acetate Sterile water
24.6 g 80 ml
Mw = 82.03
362
Appendix B a. Mix with magnetic stirring. b. Adjust the pH to 4.8 with acetic acid. Sterile water to 100 ml Sterilization Sterilize in an autoclave. 2h 105C Storage In aliquots of 50 to 100 l. At rt
For some months Stock solution: 5 M AR or molecular-biology quality, to be used only for in situ PCR/RT-PCR NaCl See Appendix B1.1.
B2.19
Sodium Chloride
Reagents/Solutions Sodium chloride Sterile water Precaution None Protocol Sodium chloride Sterile water Sterilization Sterilize in an autoclave. Storage
14.6 g to 50 ml 2h 105C At rt
Mw = 58.44
For some months Stock solution: 10 N RP quality NaOH See Appendix B1.1.
B2.20
Sodium Hydroxide
Reagents/Solutions Sodium hydroxide Sterile water Precaution Extremely corrosive reagent Protocol Sodium hydroxide Sterile water Mix slowly. Storage
40 g to 100 ml At rt
Mw = 40.00
B2.21
Tris
Stock solution: 1 M
Reagents/Solutions 10 N hydrochloric acid Sterile water Trishydroxymethylaminomethane Precaution None
See Appendix B1.1. C4H11NO3, Tris base in powder
363
Reagents Protocol Dissolve: Tris base Sterile water Storage
121 g to 1 l At rt
Mw = 121.16 Some weeks Stock solution: 0.1 to 0.3% AR quality See Appendix B1.1. C34H62O11
B2.22
Triton X-100
Reagents/Solutions Sterile water Triton X-100 Precaution Reagent difcult to pipette Protocol Dissolve: Triton X-100 Sterile water Storage
0.10.3 g to 100 ml At rt
Mw = 646.87 Difcult to pipette with exactitude Some weeks
B3
B3.1
BUFFERS
Acetylation Buffer
100 mM Triethanolamine buffer; pH 8.0 RP quality See Appendix B1.1. C6H15NO3 (Tris (hydroxy-2-ethyl)-amine)
Reagents/Solutions 10 N hydrochloric acid Sterile water Triethanolamine Precaution Toxic Protocol a. Mix: Triethanolamine Sterile water b. Adjust pH: HCl Sterile water Storage
3.32 ml 200 ml pH 8.0 1 ml to 250 ml At 4C
Mw = 149.19
Some hours 200 mM Cacodylate buffer; pH 7.4 AR quality Na(CH3)2AsO23H2O See Appendix B1.1.
B3.2
Cacodylate Buffer
Products/Solutions N hydrochloric acid Sodium cacodylate Sterile water Precaution None 364
Appendix B Protocol a. Dissolve: Sodium cacodylate Sterile water b. Check the pH. Sterilization Autoclave. Storage
4.28 g to 100 ml 7.4 2h 105C At 4C
Mw = 214.05
Some days Stock solution: 10X RP quality BSA 5X, crystallized See Appendix B2.12. See Appendix B1.1. See Appendix B2.21.
B3.3
DNase I Buffer
Reagents/Solutions Bovine serum albumin 10 N hydrochloric acid 500 mM magnesium chloride Sterile water 1 M Tris Precaution Risk of RNase contamination; prepare under sterile conditions Protocol TrisHCl; pH 7.6 500 mM MgCl2 100 mM BSA 0.5 mg/ml Storage In aliquots At 20C
Some hours
B3.4
B3.4.1
Phosphate Buffer
1 M Phosphate Stock solution AR quality Na2HPO4. Hydrated reagents can be used, taking into account variations in molar mass. NaH2PO4H2O See Appendix B1.1.
Reagents/Solutions Disodium phosphate Monosodium phosphate Sterile water Precaution Preparations obtained in sachets should be transferred to sterile containers, with sterile water. Protocol a. Dissolve: Disodium phosphate 14.19 g Monosodium phosphate 13.8 g Sterile water to 100 ml b. Check pH. pH 7.4 Sterilization Autoclave. 2h 105C Storage At rt or 4C
Mw = 141.96 Mw = 138.00
Some weeks 365
Reagents B3.4.2 Phosphate/NaCl 100 mM phosphate buffer/300 mM NaCl; pH 7.4 AR quality See Appendix B3.4.1. See Appendix B2.19. See Appendix B1.1.
Products/Solutions 1 M phosphate buffer 5 M sodium chloride Sterile water Precaution None Protocol a. Add: 5 M sodium chloride 1 M phosphate buffer Sterile water b. Check pH. Sterilization Autoclave. Storage B3.4.3 PBS
6 ml 10 ml to 100 ml pH 7.4 2h 105C At rt or 4C
Some days Phosphate buffer saline 10 mM mono-di-phosphate buffer/150 mM NaCl/10 mM KCl; pH 7.4 Can be prepared in stock solution 10X AR quality, and to be used only for in situ PCR/RT-PCR Na2HPO412H2O KH2PO43H2O KCl NaCl See Appendix B2.19. See Appendix B2.20. See Appendix B1.1.
Reagents/Solutions Disodium phosphate Monopotassium phosphate Potassium chloride Sodium chloride
10 N sodium hydroxide Sterile water Precaution Risks of contamination Protocol a. Add: Sodium chloride 8.76 g Potassium chloride 0.74 g Disodium phosphate 3.58 g Monopotassium phosphate 1.90 g Sterile water to 1 l b. Adjust the pH with sodium hydroxide. 7.4 Sterilization Sterilize in an autoclave. 2h 105C Storage At rt up to opening then at 4C
Mw = 58.44 Mw = 74.56 Mw = 358.14 Mw = 190.15
Some weeks Some hours
366
Appendix B
B3.5
SSC Buffer
Standard Saline Citrate buffer Stock solution: 20X 3 M sodium chloride; 300 mM sodium citrate; pH 7.0 RP quality See Appendix B2.20. NaCl C6H5Na3O72H2O See Appendix B1.1.
Reagents/Solutions 10 N sodium hydroxide Sodium chloride Sodium citrate Sterile water Precaution None Protocol a. Add: Sodium chloride Sodium citrate Sterile water b. Adjust the pH with sodium hydroxide. Sterilization Autoclave. Storage
175.3 g 88.2 g to 1 l 7.0
Mw = 58.44 Mw = 294.10
2h 105C At rt prior to opening then at 4C
Some weeks Some hours Tris/EDTA AR quality, to be used only for in situ PCR/RT-PCR C10H14N2O8Na22H2O (ethylene diamine tetra-acetic acid, or Titriplex III) C4H11NO3 Stock solution: 10X 100 mM TrisHCl/10 mM EDTA; pH 7.6 C10H14N2O8Na22H2O or 500 mM stock solution (see Appendix B2.13) See Appendix B1.1. C4H11NO3 or 1 M stock solution (see Appendix B2.22)
B3.6
TE (TrisEDTA) Buffer
Reagents EDTA Tris (hydroxymethylaminomethane) B3.6.1 TE buffer
Reagents/Solutions EDTA 10 N hydrochloric acid Sterile water Tris Precaution Toxicity of EDTA
367
Reagents Protocol a. Add: Tris EDTA b. Adjust the pH with HCl. Sterile water Sterilization Sterilize in an autoclave. Storage
12.1 g 3.7 g 7.6 to 1 l
Mw = 121.16 Mw = 372.24
2h 105C At rt up to opening then at 4C
Some weeks Some days Stock solution: 10X 100 mM TrisHCl/10 mM EDTA/5 M NaCl; pH 7.6 AR quality NaCl See Appendix B1.1. See Appendix B3.6.1.
B3.6.2
TENaCl buffer
Products/Solutions 10 N hydrochloric acid Sodium chloride Sterile water 10X TE buffer Precaution Toxicity of EDTA Protocol a. Add: Sodium chloride 10X TE buffer b. Adjust the pH with HCl. Sterilization Autoclave. Storage
29.2 g to 100 ml 7.6 2h 105C At rt until opened then at 4C
Mw = 58.44
Some weeks Some hours
B3.7
B3.7.1
TrisHCl Buffer
TrisHCl buffer Solution: 100 mM RP quality See Appendix B1.1. See Appendix B2.21.
Reagents/Solutions 10 N hydrochloric acid Sterile water 1 M Tris Precaution Risk of contamination by exogenous organisms Protocol a. Add: Tris 1 vol Sterile water 9 vol
368
Appendix B b. Adjust the pH with HCl: pH pH Sterilization Storage B3.7.2 TrisHCl/CaCl2 buffer
7.6 8.5 At 4C Very difcult, if not impossible Some days 20 mM TrisHCl/2 mM CaCl2; pH 7.6 RP quality See Appendix B2.3. See Appendix B1.1. See Appendix B2.21.
Reagents/Solutions 1 M calcium chloride 10 N hydrochloric acid Sterile water 1 M Tris Precaution Risk of contamination Protocol a. Add: Tris Calcium chloride b. Adjust the pH with HCl: Sterile water Sterilization Storage B3.7.3 TrisHCl/glycine buffer
20 ml 2 ml 7.6 to 1 l At 4C Very difcult, if not impossible Some days 50 mM TrisHCl/50 mM glycine; pH 7.4 RP quality C2H5NO2 See Appendix B1.1. See Appendix B2.21.
Reagents/Solutions Glycine 10 N hydrochloric acid Sterile water 1 M Tris Precaution Risk of bacterial contamination Protocol a. Add: Glycine Tris Sterile water b. Adjust the pH with HCl Sterilization Storage In aliquots
0.37 g 5 ml to 100 ml 7.4 At 20C At 4C
Mw = 75.07
Very difcult, if not impossible Some weeks Some hours 10 mM TrisHCl/5 mM MgCl2; pH 7.3 (dilution buffer for DNase) RP quality See Appendix B2.12. See Appendix B1.1. See Appendix B2.21. 369
B3.7.4
TrisHCl/MgCl2 buffer
Reagents/Solutions 10 N hydrochloric acid 1 M magnesium chloride Sterile water 1 M Tris
Reagents Precaution Risk of bacterial contamination Protocol a. Add: 1 M Tris 1 M MgCl2 b. Adjust the pH with HCl: Sterile water Storage In aliquots
1 ml 500 l 7.3 to 100 ml At 20C At 4C Some weeks Some hours 20 mM TrisHCl/300 mM NaCl; pH 7.6 Visualization buffer RP quality See Appendix B2.19. See Appendix B1.1. See Appendix B2.21.
B3.7.5
TrisHCl/NaCl buffer
Reagents/Solutions 10 N hydrochloric acid 5 M sodium chloride Sterile water 1 M Tris Precaution Risk of bacterial contamination Protocol a. Add: 5 M sodium chloride 1 M Tris Sterile water b. Adjust the pH with HCl. Storage Prepare just before use. B3.7.6
6 ml 2 ml to 100 ml 7.6
TrisHCl/NaCl/MgCl2 buffer
20 mM TrisHCl/300 mM NaCl/50 mM MgCl2 RP quality See Appendix B2.12. See Appendix B2.19. See Appendix B1.1. See Appendix B2.21.
Reagents/Solutions 10 N hydrochloric acid 1 M magnesium chloride 5 M sodium chloride Sterile water 1 M Tris Precaution Risk of bacterial contamination Protocol a. Add: 1 M Tris 5 M NaCl 1 M MgCl2 b. Adjust the pH with HCl: pH pH Sterile water 370
20 ml 60 ml 50 ml 7.6 9.5 to 1 l Visualization of peroxidase Visualization of alkaline phosphatase
Appendix B Storage Prepare extemporaneously.
B4 FIXATIVES
B4.1
B4.1.1
Formol
Neutral buffered formalin
Solution stock
Reagents/Solutions Na2HPO4 NaH2PO4H2O Formaldehyde 37 to 40% Distilled water Precaution Risks for the eyes and the respiratory passages Protocol a. Dissolve Na2HPO4 6.5 g NaH2PO4 H2O 4g b. Add: Distilled water and 100 ml 500 ml 40% formaldehyde and make up to 1 l with distilled water B4.1.2 Formol saline
Commercial solution DEPC water (see Appendix B2.1.2) Manipulation under a hood
Check the pH and adjust if necessary to pH 7.2 using appropriate salt
Reagents/Solutions Formaldehyde 37 to 40% Distilled water Sodium chloride Precaution Risks for the eyes and the respiratory passages Protocol a. Mix 100 ml formaldehyde and 900 ml of distilled water. b. Dissolve 9 g of sodium chloride in the mixture. Storage To be avoided after dilution
Commercial solution DEPC H2O (see Appendix B2.1.2) See Appendix B2.19. Manipulation under a hood
B4.2
Glutaraldehyde (2.5%)
Solution to be prepared just before use Electron microscopy quality OCH(CH2)3CHO The higher the concentration, the stabler the solution See Appendix B3.4. 371
Products/Solutions 25 to 70% glutaraldehyde
200 mM phosphate buffer
Reagents Precaution Volatile and dangerous chemical Protocol Mix: 25% glutaraldehyde Phosphate buffer Distilled water Storage Use immediately after preparation.
Use under a fume hood Depending on the concentration of the original solution
1 ml 5 ml to 10 ml
A couple of hours at 4C
B4.3
Paraformaldehyde
Equipment Hood Magnetic stirring, with heating Reagents/Solutions Distilled water Paraformaldehyde in powder 1 M phosphate buffer 10 N sodium hydroxide B4.3.1 Paraformaldehyde 40%
RP quality See Appendix B1.1. HO(CH2O)nH In the form of nonhydrated powder See Appendix B3.4.1. See Appendix B2.20.
Stock solution
Precaution Manipulate under hood. Protocol a. Place in an Erlenmeyer: Distilled water 100 ml Paraformaldehyde 80 g b. Allow to hydrate for some minutes, then mix the solution with magnetic stirring. c. Add caustic soda until the solution clears: 0.5 ml NaOH Distilled water to 200 ml d. Close the recipient with aluminum foil. e. Heat and shake. 20 min 60C f. Filter and allow to cool. Storage In aliquots At 4C At 20C
The solution is milky in appearance.
The solution clears.
1 month Several months Do not refreeze
B4.3.2
Paraformaldehyde 4%
Precaution Manipulation under fume hood
372
Appendix B Protocol a. Place in a glass ask: Paraformaldehyde Distilled water b. Stir while heating. c. Allow to cool, and add: 1 M phosphate buffer d. Adjust the pH with NaOH Distilled water e. Filter and allow to cool. Storage To be used immediately
4g 50 ml 60C 10 ml pH 7.4 to 100 ml At 20C See Appendix B3.4.1.
Some days Do not refreeze. Working solution
B4.3.3
Paraformaldehyde 4%/ glutaraldehyde 0.05%
Reagents/Solutions Glutaraldehyde 25 to 75% 4% Paraformaldehyde (PF) in 100 mM phosphate buffer; pH 7.4 Precaution Manipulation under hood Protocol 4% buffered paraformaldehyde 200 ml Glutaraldehyde 133400 l Storage Use immediately.
OCH(CH2)3CHO, microscopy quality; the higher the concentration, the stabler the solution. See Appendix B4.3.2.
According to the glutaraldehyde concentration
B5 EMBEDDING MEDIA
B5.1

Materials
Disposable pipettes Fume hood Glass containers Gloves Magnetic stirrer Mask Propipette
Or electric stirrer
373
Reagents
B5.2
B5.2.1
Epoxy Resins
Eponaraldite
Products/Solutions Araldite 502 Dodecenyl succinic anhydride Hexahydrophtalic anhydride 2,4,6-Tris-dimethylamine methyl phenol Precautions Toxic products Avoid all contact with the skin. Protocol a. Mix all the following products in a glass container: Araldite 502 20 ml DDSA 60 ml Epox 812 25 ml b. Add the hardener to the mixture: Eponaraldite mixture 20 ml DMP 30 300 l
DDSA, hardener Epox 812 resin DMP 30 (accelerator); very dangerous and irritant chemical Risk of developing allergies Wear a mask. Wear gloves. All traces of resin on the skin must be removed with soapy water. The mixture is very viscous.
300 l of resin is required to ll a small gelatin capsule (size 00). The amount of the product required can rise by 20% depending on how often the container is opened.
Storage Prepare just before use and keep away from light. B5.2.2 Epon DDSA, hardener Aliphatic carbon chain (mixture of glycidyl ethers and mono-trisubstituted glycerol) MNA, hardener DMP 30, accelerator; very dangerous and irritant chemical Risk of developing allergies Wear a mask. Gloves must be worn. The mixture is very viscous. A glass rod xed to an electric mixer may be used to prepare a large quantity.
Products/Solutions Dodecenyl succinic anhydride Epox 812 resin Methyl nadic anhydride 2,4,6-Tris-dimethylamine methyl phenol Precautions Toxic products Avoid all skin contact Protocol Stock solution a. Thoroughly mix all the following products in a glass container:
374
Appendix B DDSA Epox 812 MNA Solution for use b. Add the hardener to the mixture: Epon mixture DMP 30 50 ml 81 ml 44.5 ml To polymerize the resin 20 ml 300 l The amount of the product required can rise by 20% depending on how often the container is opened.
Storage The mixture without the hardener can be kept at room temperature for 1 month. The mixture with the hardener can be kept at 4C for several hours or overnight.
Time taken to impregnate the tissue.
B5.3
B5.3.1
Acrylic Resins
Lowicryl K4M
Methacrylate resin 2-hydroxyethyl-acrylate; resin sold as a kit
Products/Solutions Lowicryl K4M Solution A (Cross-linker) Solution B (monomer) Initiator C Precautions Toxic products Very toxic resin Avoid all contact with the skin Avoid inhaling the vapors
Clear liquid, distinctive smell Accelerates polymerization Risk of developing allergies An automatic embedding system is available. Methacrylate is irritant to the skin, eyes and respiratory system. Gloves and a mask must be worn. All traces of the resin on the skin must be removed with running water. Pipette using a propipette. Use a fume hood. Quantity to prepare: 1 ml/tube for impregnation 500 l/gelatin capsule for embedding The mixture is very runny. Wear a mask during weighing. Pipette using an automatic pipette. Stir gently (to avoid bubbles). 1 to 2 years The storage depends on the time taken to impregnate the samples. 375
Protocol
a. Thoroughly mix the following products in a glass container: Initiator C 0.1 g Solution B 17.30 ml b. Add Solution A 2.70 ml c. Embed the samples. Storage The different products of the kit are kept at rt in a dry, dust-free place away from heat. The mixture can be kept for up to 2 days at 30C.
Reagents Wastes Keep waste in special containers.
Do not throw down the sink. May be destroyed by incineration. London Resin Medium hardness Acrylic polyhydroxyaromatic resin A single stock solution of methacrylate Made up solely of monomers used in medicine and dentistry Less allergenic than Lowicryl K4M Recommended to wear a mask and gloves; pipette with a propipette Use in a fume hood Quantity to make up: 1 ml per tube for impregnation 0.5 ml per gelatin capsule for embedding Use in a fume hood. Use a propipette. 1 drop (optional). Very quickly with a vorex. Polymerization is very fast: less than 15 min with an accelerator, several hours without. For several months Very sensitive to the heat Do not throw down the sink. Stock solution ready to use The commercially available solution is ready to use. Always wear gloves. Always wear a mask; pipette using a propipette under a fume hood. Use a fume hood. Quantity to make up: 1 ml per tube for impregnation 0.5 ml per gelatin capsule for embedding
B5.3.2
LR White medium
Products/Solutions LR White medium Accelerator Precautions Weakly toxic and irritant resin
Avoid contact with the skin Avoid inhaling vapors Protocol
a. Take out the products just before making the mixture. b. Place in a glass container: LR White 5 ml Accelerator 5 l c. Mix immediately. 12 min d. Embed the samples very quickly. Storage Wastes Keep the waste in glass containers. B5.3.3 Unicryl At 4C
Product Unicryl Precautions Toxic resin Avoid contact with the skin. Avoid inhaling vapors. Protocol
a. Take out the stock solution just before impregnation.
376
Appendix B b. Embed biological samples at low temperature: Samples are dehydrated progressively at low temperature (from 0C down to 20C) without a substitution stage. After the last change of solvent, the stock solution is directly impregnates at 20C for several hours. Storage At 30C At 4C
Rapid protocol Easy to use and the resin is ready to use, cutting down the risk of handling For several months For 1 year
B6 REVELATION
B6.1
B6.1.1
Autoradiography
Standard developer AR quality C6H4(OH)2 [OHC6H4NH(CH3)]2H2SO4 KBr Na2CO3 Na2SO3 See Appendix B1.1.
Reagents/Solutions Hydroquinone Metol Potassium bromide Sodium carbonate Sodium sulte anhydride Sterile water Precaution Toxic reagent Working solution
To be manipulated with gloves Preferably ready-to-use solutions recommended by the manufacturer of the emulsions and lms
a. Dissolve the following components: Metol 0.5 g Hydroquinone 6g Sodium sulte 50 g Sodium carbonate 32 g Potassium bromide 2g Running water to 1 l b. Filter. Storage At 4C Protocol Pure 4 min 17C Diluted in water (1:1 v/v) 6 min 17C B6.1.2 Fixative
Anhydride Anhydride
Reagents/Solutions Distilled water Sodium thiosulfate
Na2S7O3
377
Reagents Working solution Sodium thiosulfate 30% Distilled water Protocol Working solution Storage Some weeks at room temperature
60 g 200 ml 15 min
Mw = 158.00 Minimum volume for a staining tray
B6.2
B6.2.1
Immunocytology
Blocking solutions
B6.2.1.1 NONSPECIFIC SITES Reagents/Solutions Blocking agents Bovine serum albumin (BSA) Dried skimmed milk Fish gelatin Goat serum Ovalbumin 0.01% Triton X-100 Buffers 100 mM phosphate buffer; pH 7.4 50 mM TrisHCl buffer/300 mM NaCl; pH 7.6 Precaution Do not leave at room temperature. Working solution Dilute: Blocking agents 12% Buffer to 100 ml Storage At 20C B6.2.1.2 ENDOGENOUS ALKALINE PHOSPHATASES Levamisole Reagents/Solutions Levamisol (2,3,5,6-tetrahydro-6phenylimidazole) 50 mM TrisHCl buffer/100 mM NaCl/ 50 mM MgCl2; pH 9.5 Precaution None 1 M stock solution Dissolve: 100 mM levamisol 24 mg Revelation buffer 1 ml 378
See Appendix B3.4. See Appendix B3.7.5.
Some weeks Endogenous enzyme activity may be inhibited by levamisole or by heat treatment.
C11H12N2S (2,3,5,6-ttrahydro-6-phenylimidazole) See Appendix B3.7.6.
Mw = 240.80
Appendix B 1 mM working solution Stock solution Revelation buffer Storage Use immediately after preparation.
1 l 990 l
B6.2.1.3 ENDOGENOUS PEROXIDASES Hydrogen peroxide/buffer Reagents/Solutions 30% hydrogen peroxide 50 mM TrisHCl/300 mM NaCl; pH 7.6 Precaution Avoid contact with the skin. Solution for use Mix: Hydrogen peroxide Buffer Storage Use immediately after preparation. Hydrogen peroxide/methanol Reagents/Solutions 30% hydrogen peroxide 100% methanol Precaution Contact dangerous Working solution Mix: Hydrogen peroxide Methanol Storage Use immediately after preparation. B6.2.2 Chromogens
110 V See Appendix B3.7.5, or use a phosphate buffer (see Appendix B3.4). Wash in running water
3 ml 100 ml
110 V
3 ml 100 ml
B6.2.2.1 ALKALINE PHOSPHATASE NBT-BCIP
There are other chromogen substrates that give different color precipitates (i.e., Fast-Red). Use a commercial solution. (CH3)2NOCH C40H30Cl2N10O6 C8H6NO4BrCIPC7H9N See Appendix B3.7.6.
Reagents/Solutions Dimethylformamide Substrates NBT (Nitroblue tetrazolium) BCIP (5-bromo-4-chloro-3-indolyl phosphate) TrisHCl/NaCl/MgCl2 buffer; pH 9.5 Precaution Avoid contact with the skin. Use immediately after preparation and keep out of the light.
379
Reagents Working solution Mix: NBT
BCIP
Visualization buffer Storage Reagents in commercial solutions are stored at 4C. B6.2.2.2 PEROXIDASE Diaminobenzidine tetrahydro-chloride Inhibition of peroxidase activities There are other chromogen substrates that give different color precipitates (i.e., 4-chloro1-naphthol or 3-amino-9-ethylcarbazole). C12H14N44HCl 110 vol See Appendix B3. Tablet form preferable Prepare extemporaneously in darkness Mw = 360.12 Tablet of DAB to be dissolved in water
0.34 mg or 4.5 l 0.18 mg or 3.5 l to 1 ml
Mw = 817.70 75 mg/ml dimethylformamide Mw = 433.60 50 mg/ml dimethylformamide
Reagents/Solutions DAB 30% hydrogen peroxide TrisHCl/NaCl buffer; pH 7.6 Precaution Reagent dangerous if inhaled Working solution a. Dissolve: DAB or 1 tablet 10 mg Revelation buffer 10 ml b. Filter the solution. c. To the ltrate, add: 3% hydrogen peroxide/H2O 10 l Storage At 20C, in aliquots, without hydrogen peroxide
Commercial solutions (DAB and H2O2) can be stored at 4C.
B7 STAINING/COATING
B7.1
B7.1.1
Light Microscopy
Cresyl violet
Reagents/Solutions Cresyl violet Distilled water Precaution Filter the solution before use.
380
Appendix B Working solution Dissolve Cresyl violet Distilled water Storage B7.1.2 Eosin
1g 100 ml At rt
For some weeks
Reagents/Solutions Distilled water Eosin Precaution None Working solution Dissolve Eosin Distilled water Storage
5g 100 ml At rt
In an opaque container For several months
B7.1.3
Harriss hematoxylin C2H4O2 Aluminum potassium sulfate (AlK(SO4)2 12H2O) C16H14O6
Reagents/Solutions Acetic acid Alum Distilled water Ethyl alcohol Harriss hematoxylin Yellow mercury oxide Precautions Mercury oxide is toxic. Filter the solution before use. The solution matures with time. Working solution a. Dissolve: Hematoxylin 5g Ethyl alcohol 50 ml b. Dissolve while warm: Alum 100 g Distilled water 1l c. Mix a and b d. Heat to boiling point, and add: Mercury oxide 2.5 g e. Withdraw rapidly from the heat, and add: Acetic acid 20 ml Storage At rt
This increases in its dyeing properties. Mw = 302.30 Mw = 474.39
Mw = 60.05 For some months In an opaque container
381
Reagents B7.1.4 Methylene green
Reagents/Solutions Distilled water Methyl green Precaution Filter before use. Working solution Dissolve: Methyl green Distilled water Storage B7.1.5 Rapid nuclear red
C27H35N3BrClZnCl2
1g 100 ml At rt
Mw = 653.2 For some weeks Not to be confused with Fast-Red Al2(SO4)3 C14H9NO7S
Reagents/Solutions Aluminum sulfate Distilled water Rapid nuclear red Precaution Filter the solution before use. Working solution a. Prepare 1 l of aluminum sulfate at 5%: Aluminum sulfate 50 g Distilled water 1l b. Heat to boiling point. c. Withdraw from the heat, and add: Nuclear red 1g Storage At 4C B7.1.6 Toluidine blue
Mw = 342.1
Mw = 335.3 For some weeks 1% aqueous toluidine blue
Reagents/Solutions Distilled water Sodium tetraborate Toluidine blue Precaution Filter the solution before use. Stock solution a. Mix and dissolve at room temperature: Toluidine blue 1g Sodium tetraborate 1g Distilled water 100 ml b. Filter. Working solution Dilute: Stock solution 1 vol Distilled water 50 vol Storage At rt
Na2B4O712H2O C15H16N3SCl
Mw = 305.80 Mw = 381.37
For some months Filter before use
382
Appendix B
B7.2
B7.2.1
Electron Microscopy
Uranyl acetate Electron-microscopy quality (EM) NH4OH (10% solution) C2O4H22H2O See Appendix B1.1. (CH3COO)2 UO22H2O Use a fume hood. Emits radiations
Products Ammonia 95% ethanol Oxalic acid Sterile distilled water Uranyl acetate Precautions Uranium is radioactive. Avoid all contact with the skin. Avoid inhaling uranyl powder.
Wear a mask while weighing out the uranyl acetate.
B7.2.1.1 2.5% ALCOHOLIC URANYL ACETATE Protocol a. Prepare just before use in a brown glass container: 5% aqueous uranyl acetate 1 vol 95% alcohol 1 vol b. Filter in the dark. c. Stain in the dark. Storage Do not store. B7.2.1.2 2 TO 5% AQUEOUS URANYL ACETATE Protocol Uranyl acetate 0.51.25 g Distilled water Mix. Filter using a Millipore. Storage B7.2.1.3 25 ml 0.22 m At 4C
In a dark room with a safe light Saturated solution (see Appendix B7.2.1.2) Alcoholic uranyl is very sensitive to the light.
Stock solution Dissolve at room temperature in the dark with a magnetic stirrer for 1 to 2 h.
For 1 month away from light 4% aqueous uranyl acetate/300 mM oxalic acid; pH 7.5 Electron microscopy quality See Appendix B7.2.1.2.
4% NEUTRAL URANYL ACETATE
Solutions 4% aqueous uranyl acetate 10% ammonia 300 mM oxalic acid Precaution Away from the light Protocol a. Mix: 4% aqueous uranyl acetate 300 mM oxalic acid
1 vol 1 vol 383
Reagents b. Adjust the pH 10% ammonia c. Filter using a Millipore. Storage B7.2.2 Lead citrate
77.5 0.22 m At 4C
Use pH paper; uranyl salts cause deterioration of the elecrodes. For 1 month According to Reynolds Electron microscopy quality Pb(NO3)2 C6H5Na3O72H2O NaOH
Products Lead nitrate Sodium citrate Sodium hydroxide Solutions 1 N sodium hydroxide Distilled water Protocol Sodium citrate
See Appendix B2.20. See Appendix B1.1. 0.882 g Mw = 294.10 1% sodium tartrate can also be used. Mw = 331.23 A homogenous precipitate forms. Mw = 40.00 The precipitate dissolves at a basic pH 10.
Lead nitrate 0.662 g a. Dissolve the two products separately in distilled water. b. Mix the two solutions thoroughly. c. Add: 1 N sodium hydroxide 4 ml d. Mix until the precipitate dissolves completely. e. Add: Distilled water to 25 ml f. Filter with a 0.22 m Millipore lter. Storage In aliquots At 4C
For 1 month Fill the aliquots to the top to avoid contact with carbon dioxide in the air.
B7.2.3
Methylcellulose Stock solution Electron microscopy quality Tylose is the commercial name of methyl cellulose. See Appendix B1.1.
B7.2.3.1 2% METHYLCELLULOSE Products Methylcellulose (Tylose) Solution Distilled water Precaution Do not stir. Take the quantity needed from the top of the tube. Protocol Methylcellulose 2g Distilled water 100 ml
384
Appendix B a. Heat. b. Place with stirring. c. Place without stirring until the Tylose has dissolved. d. Centrifuge. Storage 95C 15 min 4C overnight 1530 days 4C 100,000 g 60 min At 4C At 20C
Make up stocks and keep at 4C. The solution forms very slowly (>1 month). Not indispensable For several weeks Solution in use For several months Reserve solution
B7.2.3.2 0.8% METHYLCELLULOSE, 0.2%

NEUTRAL URANYL ACETATE
A staining solution can also be used for coating sections of frozen tissue. See Appendix B1.1. See Appendix B7.2.1.3. See Appendix B7.2.3.2.
Solutions Distilled water 2% neutral uranyl acetate Methylcellulose 2% Precaution None Protocol a. Add the methylcellulose and the neutral uranyl acetate to give a nal concentration of 0.2%: Neutral uranyl acetate 200 l Tylose 800 l Distilled water 100 ml b. Stir. c. Centrifuge. Storage At 4C At 20C B7.2.4 Sodium silicotungstate (0.5%)
Or more, if necessary Eliminates bubbles For several weeks Solution for use For several months
Solutions Distilled water Sodium silicotungstate Precaution Avoid all contact with the skin. Protocol Dissolve in distilled water: Sodium silicotungstate Distilled water Storage
See Appendix B1.1. AR quality
0.5 g 100 l At 4C
For several weeks
385
Reagents
B8
B8.1
MOUNTING MEDIA
Aqueous
Aqueous mounts are used where the reaction precipitate is soluble in alcohol: Buffered glycerine Commercial media Moviol B8.1.1 Buffered glycerine
See Appendix B8.1.1. See Appendix B8.1.2.
Reagents/Solutions Glycerol 100 mM phosphate buffer, or PBS Precaution None Preparation Glycerol 1 vol Buffer 1 vol Storage At 4C B8.1.2 Moviol
See Appendix B3.4.2 or B3.4.3.
For some days
Reagents/Solutions Distilled water Glycerol anhydride Moviol 488 100 mM Tris-HCl buffer; pH 8.5 Precaution None Preparation a. Mix: Moviol 488 Glycerol b. Add: Distilled water c. Mix and let stand. d. Add: Buffer e. Incubate. f. Mix, centrifuge. Storage In aliquots
RP quality C3H8O3 See Appendix B3.7.1.
2.4 g 4.8 ml 6 ml 248 h 60C
Mw = 92.10
12 ml overnight 60C 5,000 g 15 min At 20C For some days
386
Appendix B
B8.2
Permanent
Permanent mounting media are used after dehydration and passage through xylene (or a similar chemical). Reagents Commercial media Precautions Toxic on contact or by inhalation Polymerization on drying Storage At room temperature in tightly closed containers
Aromatic solvents, or substitutes Xylene can be used to make the solution more uid.
387
Glossary
Glossary
A
Adenine A puric base that is found in nucleosides, nucleotides, coenzymes, and nucleic acids. It forms two hydrogen bonds with thymine and uracil in double-stranded nucleic acids. A phosphatase extracted from calf intestine (Mw = 80 kDa). Its optimum pH is alkaline. A glycoprotein produced in response to the presence of an antigen. It can combine with the antigen that induced its production. An antibody that is specic to an epitope produced by a cell culture resulting from the fusion of a tumoral cell and an antibodyproducing cell. All the immunoglobulins contained in serum from an animal immunized with a given antigen. Polyclonal antibodies recognize several epitopes (or antigenic determinants). An antibody that is specic to the antigen being sought in an immunocytological reaction. An antibody used for indirect immunological reactions, whether conjugated or not, and directed against the animal species in which the antibody IgGs were produced. Any substance that is foreign to a receptor organism. It is said to be immunogenic when it induces an immune response (which can bring about the production of antibodies). See Epitope. A serum containing an immunoglobulin that is specic to an injected protein. An epoxy resin made up of chains of aromatic glycerol polyarylether rings. Background noise, or nonspecic reactions. Adenosine triphosphatase (an enzyme that catalyzes ATP hydrolysis). A device that sterilizes objects by the use of pressurized steam. This apparatus automatically dehydrates, impregnates, and polymerizes biological samples embedded in hydrophilic resin at low temperature. A method for detecting hybrids containing isotopes.
Alkaline phosphatase Antibody
Monoclonal
Polyclonal
Primary Secondary
Antigen
Antigenic determinant Antiserum Araldite Artifact ATPase Autoclave Automatic freeze-substitution system (AFS)
Autoradiography/radioautography
391
Glossary Activity The number of disintegrations per second in a radioactive source. The unit is the Becquerel (Bq), which corresponds to one disintegration per second. The previous unit was 11 the curie (Ci). (1 Bq = 2.7 10 Ci; 1 Ci = 7 3.7 10 disintegrations/second). The number of silver grains obtained by radiation. The distance between the location of a silver grain and a radiation source. It is dened by the energy of the isotope and the autoradiographic system. A glycoprotein (Mw = 16,400) produced in the uterus of the chicken. It contains four subunits, each of which binds a molecule of biotin 15 1 with a very high afnity (Ka = 10 M ).
Efciency Resolution
Avidin
B
Background noise Base A nonspecic signal (artifact). Nucleic acids contain two groups of bases: purines and pyrimidines. A base is a at, cyclical molecule composed of hydrogen, carbon, and nitrogen. The initials that identify the genetic code are A (adenine), G (guanine), C (cytosine), T (thymine), and U (uracil). The puric bases are guanine and adenine, whose chemical structure resembles that of the pyrimidines, combined with a pentagonal structure (the pyrimidic ring + the imidazol ring). The pyrimidic bases are cytosine and thymine (DNA) or uracil (RNA), whose chemical structure is hexagonal. A parameter whereby the length of a double strand of DNA can be determined. A base consists of a nucleotide, and a base pair is a combination of two nucleotides, one on each of the two complementary DNA strands. A microscope whose object is illuminated directly and intensely, giving an image on a light-colored background.
Puric
Pyrimidic
Base pair
Bright-eld microscope
392
Glossary
C
Carbon lm Cellular clone Carbon vaporized onto grids covered with collodion or formvar. All the cells that are produced by the division of a given mother cell (and which are thus genetically identical). An agent that captures bivalent cations 2+ (e.g., Ca ), thus blocking the action of enzymes (e.g., EDTA). The appearance of background noise in a photographic emulsion due to the chemical effect of a biological preparation. A component that produces a colored substance through an enzymatic reaction. From the Greek khroma, meaning color, and soma, meaning body. It contains most or all of the cellular DNA, and thus the genetic information. A DNA molecule that is capable of replicating itself (replicon). It is used to transport a piece of foreign DNA (e.g., a gene) into a receptor cell. It can be a plasmid, a phage, etc. A step that consists of placing a layer of saccharose or methylcellulose on a tissue sample or a section. A sequence of three bases (or triplets) in mRNA. It codes for an amino acid, and can also indicate the beginning or the end of a transcription process. A codon that initiates protein synthesis. There is only one such codon, i.e., AUG, which also codes for methionine. Or stop: a codon that does not code for an amino acid, but acts as a signal for the termination of a protein synthesis operation (e.g., UAA, UAG, UGA). An organic lm used as a membrane support on a grid. Celloidin, also known as collodion, is a puried form of nitrocellulose. Light-microscopic staining that is used to obtain a different contrast from that of the hybridization signal, such that the general tissue structure is dened. The transformation of water in cells from a liquid to a solid state, with the stopping of cell functioning and the hardening of the sample.
Chelator
Chemography
Chromogen Chromosome
Cloning vector
Coating
Codon
Initiation
Termination
Collodion
Counterstaining
Cryoxation
393
Glossary Cryopolymerization Cryoprotectant The hardening of a resin by ultraviolet radiation at low temperature. A molecule that, when added to a sample in the aqueous phase, allows it to be frozen in an amorphous state. The incubation of samples in a solution that limits crystallization during the freezing process. A section of frozen tissue. The penetration, at low temperature, of samples xed in solvents, then in solvent resin mixtures of increasing resin concentration, and nally in pure resin. An apparatus for cutting semithin and ultrathin sections of frozen tissue. A technique for cutting semithin and ultrathin sections of frozen tissue. A pyrimidic base that is a component of nucleosides, nucleotides, and nucleic acids.
Cryoprotection
Cryosection Cryosubstitution
Cryoultramicrotome Cryoultramicrotomy Cytosine
D
Dehydration Denaturation The progressive replacement of cell water through successive baths of increasing concentrations of solvent (e.g., ethanol or acetone). The separation of the two strands of a DNA double helix, either by heat or by the action of chemicals (with a change of conformation of the nucleic acids). It can also be applied to certain types of RNA. A mixture of polymers that, when added to the hybridization medium, reduces the background noise. Nucleic acid that is found in the nucleus of the cell (and which, in all cellular organisms, carries genetic information). The DNA molecule is a polynucleotide composed essentially of deoxyribonucleotides linked by phosphodiester bonds. A DNA copy of an RNA molecule. Generally messenger RNA. An enzyme in which two fragments of DNA are combined via the formation of a new phosphodiester bond.
Denhardts solution
Deoxyribonucleic acid (DNA)
Complementary (cDNA) Ligase
394
Glossary Polymerase Deoxynucleoside Deoxynucleotide Deoxyribose Deproteinization An enzyme that synthesizes a DNA molecule from a DNA template. A molecule composed of a base and a deoxyribose. See Nucleotide. A ribose that has lost an oxygen atom at 2. An enzymatic treatment that partly eliminates proteins, and in particular those associated with nucleic acids. A macromolecule, generally in the form of a sulfate (dextran sulfate), which, when added to a hybridization buffer, increases the effectiveness of the hybridization process by concentrating the probe. A tool with an extrane point (for delicate work), which facilitates the positioning of semithin or ultrathin sections. See Deoxyribonucleic acid An enzyme that destroys DNA in a specic way. A process whereby a DNA molecule copies itself exactly, either in vivo or in vitro.
Dextran
Dissecting needle
DNA DNase Duplication
E
Electron microscope The type of microscope that is used to observe ultrathin tissue sections. Sections are placed on a grid and bombarded by a beam of electrons, which are deected by substances of high atomic weights, thus giving rise to contrasts. A technique used to detect and quantify specic antibodies or antigens. The different steps (dehydration, impregnation, polymerization) leading to obtaining a block from which ultrathin sections of a sample can be cut. Energy released by the emission of radiation, expressed in MeV (mega-electronvolts). A molecule that is present in a biological sample. A protein that catalyzes a specic chemical reaction. Its name generally suggests its function, with the sufx -ase added to the name of the molecule upon which it acts (e.g., ribonuclease cuts RNA molecules, and protease hydrolyzes proteins).
ELISA (enzyme-linked immunosorbent assay) test Embedding
Emission energy Endogen Enzyme
395
Glossary Epitope Also known as antigenic determinant. It is the part of an antigen that is recognized by an antibody (an epitope is a structure that can combine with a single antibody molecule). An antigen generally has several epitopes, each corresponding to a sequence of three to six contiguous or noncontiguous amino acids. An epoxy resin. See Resin. A contaminant (in laboratory equipment, etc). A transcribed or coding sequence in a gene.
Epon Epoxy Exogen Exon
F
Fab An antigen-binding fragment obtained by using an enzyme (papain) to cut an antibody. Enzymatic cutting takes place upstream from the disulde cross-links that exist between the two heavy immunoglobulin chains (Mw = 45 kDa). A fragment obtained by using an enzyme (pepsin) to cut an antibody. It represents two antibody sites (Mw = 100 kDa). A fragment that has no antibody activity, but that crystallizes easily. It is specic to the species that was the source of the immunoglobulins (Mw = 50 kDa). Its purpose is to stabilize cell metabolism and deactivate endogenous RNase, while conserving cell morphology and the integrity of nucleic acids. A chemical that favors the denaturing of nucleic acids. The addition of 1% formamide to the hybridization solution lowers the hybridization temperature by 0.65C for DNA and 0.72C for RNA. A strong organic lm that serves as a membrane support on a grid before ultrathin sections are placed on slides. The transition from the liquid state to the solid state. The temperature of a liquidsolid mixture. The temperature at which a sample freezes.
F(ab )2
Fc
Fixation
Formamide
Formvar
Freezing Freezing temperature
396
Glossary
G
Gene The unit of genetic information that codes for a polypeptide or a molecule of rRNA or tRNA. The complete set of genes is known as the genome. The various techniques used to modify an organisms genetic information by changing its genomic nucleic acids. The complete set of genes of a cell or a virus. The sum of the information contained in the genes of a given organism. A reticulating xative that gives rise to cross-links between different cell and tissue structures. A puric base that is found in nucleosides, nucleotides, and nucleic acids.
Genetic engineering
Genome Genotype Glutaraldehyde
Guanine
H
Half-life of a radioelement Hapten The time required for the radioactivity of an isotope to decline by half. A molecule that, although non-immunogenic, can induce the production of antibodies directed against itself when coupled to a macromolecular carrier. The labeling of nuclear probes by haptens, which are introduced either by chemical reactions or enzymes. Proteins that are rich in arginine and lysine, associated with DNA in eukaryotic chromosomes. The formation of hybrid molecules: doublestranded DNA, DNARNA, or RNARNA. As long as the sequences are complementary, such hybrids are stable. Describes a polar substance that has a strong afnity for water, or that dissolves easily in water. Describes a nonpolar substance that has no afnity for water, or that does not dissolve easily in water.
Haptenization
Histones
Hybridization of nucleic acids
Hydrophilic
Hydrophobic
397
Glossary
I
Immunocytochemistry Immunocytology A science that provides cytological evidence of antigenic constituents by means of immunochemical reactions. A science that provides cytological evidence of antigenic constituents by means of immunocytological reactions. See Antibody. A class of immunoglobulins that are predominant in serum (>85% of the immunoglobulins in serum) (Mw = 150 kDa). A class of serous immunoglobulins (Mw = 900 kDa) made up of heavy chains and light chains such as IgGs. They take the shape of a ve-pointed star with a central ring. Or substitution. The gradual replacement, in tissue, of one uid by another (e.g., alcohol by resin). A noncoding intercalary sequence of an interrupted gene that is transcribed into heterogeneous nuclear RNA (hnRNA, or primary transcripts of DNA), but is cut out during the splicing that brings about the maturation of mRNA. One of two or more forms of a given element that have different atomic weights but similar chemical properties.
Immunoglobulin G immunoglobulins (IgG)
M immunoglobulins (IgM)
Impregnation
Intron
Isotope
K
Knifemaker An apparatus for producing glass knives of the kind that are used to make frozen and/or resin-embedded tissue sections.
L
Lowicryls These resins are composed of a mixture of acrylate and methacrylate monomers. Their advantage is that they remain highly uid at low temperatures. Polymerization at 20C, or 80C, under ultraviolet radiation (360 nm). Or polar. These resins all have the same viscosity. Polymerization at 20 or 35C. Polymerization at 60C.
Hydrophilic Lowicryl K4 M Lowicryl K11 M 398
Glossary Hydrophobic Lowicryl HM 20 Lowicryl HM 23 LR White (London Resin Gold) Or nonpolar. Polymerization at 40C. Polymerization at 80C. An acrylic resin composed of a mixture of methacrylate and a hardener, specially made to be highly hydrophilic. Polymerization may take place in two ways (at 4C or 50C), depending on the required degree of hardness.
M
Melting temperature (Tm) Mer Methacrylate The temperature at which 50% of doublestranded DNA separates out into single strands. A unit of nucleic acid that can be a nucleotide or a pair of nucleotides. A monomer of aromatic polyhydroxyl acrylic resin.
N
Nanometer (nm) Nonspecicity 1/1000 micrometer, or 10 meter. Mismatching: either nonhomogeneous (probe-noncomplementary nucleic acid) or heterogeneous (protein-nucleic acid). Detection by hybridization of specic RNA fragments transferred onto a membrane. A macromolecule that carries genetic information. There are two types of nucleic acid: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids that can be used as probes. Nucleic acids that are present in a cell, but that do not belong to its genome or take part in its expression. Origin: viral, bacterial, or fungus. Nucleic acids that contain the genetic characteristics of a cell. Nucleic acids that are present in mitochondria. Nucleic acids that are sought in a cell. A molecule made up of a puric or pyrimidic base and a sugar (ribose or deoxyribose).
9
Northern blot technique Nucleic acid
Complementary Exogenous
Genomic Mitochondrial Targets Nucleoside
399
Glossary Nucleosome A DNA molecule associated with basic proteins (histones) in the eukaryotic nuclear matrix. Histones participate in the formation of chromatin, which consists of a exible chain of repeated units, namely, the nucleosomes. A nucleoside + one or more phosphate groups.
Nucleotide
O
Oligonucleotide From the Greek oligos, meaning small. Oligonucleotides are short fragments of DNA, with 15 to 60 nucleotides. The term is generally used for syntheses. A highly reductive chemical xative (sometimes incorrectly called osmic acid).
Osmium tetroxide (OsO4)
P
Palindrome Peptide Phenotype A complementary sequence contained in a given strand of DNA, forming an intrachain hybrid. A short chain of amino acids. A set of characteristics that can be identied by experimentation. The physical expression of a genotype. An enzyme that hydrolyzes phosphate groups in molecules. The addition of a phosphate group by the action of an enzyme. Progressive lowering temperature, according to the progressive low-temperature dehydration method, using liquid nitrogen vapor. A repetitive polynucleotide (A) sequence that is attached by the action of a ligase to the 3 end of a transcribed mRNA molecule. This is a characteristic property of mRNA. A synthesization enzyme. For DNA. For RNA. Able to withstand high temperatures. The amplication of a given DNA sequence by repeated cycles of denaturation, hybridization, and elongation.
Phosphatase Phosphorylation PLT
Poly (A)
Polymerase DNA polymerase RNA polymerase Thermostable Polymerase chain reaction (PCR)
400
Glossary Polymerization A process during which resin changes from its original liquid state to a solid state under the inuence of factors such as catalysts, heat, and ultraviolet radiation. An apparatus that uses ultraviolet radiation to harden resin. A chain made up of several nucleotides. RNA is a polynucleotide. The kinase enzyme. It is extracted from calf thymus, and is used in the radioactive labeling of oligonucleotide probes by phosphorylating the 5 end, with a phosphate group in the position. A complementary xation step. A reaction that brings about the insolubilization of a nucleic acid using alcohol and salt. The incubation of sections in a hybridization solution without a probe. A step that is carried out before hybridization. An oligonucleotide sequence that acts as the starting point for the neosynthesis of nucleic acids through the action of DNA polymerase. A complementary sequence at the 3 end of the sequence being studied. A sequence that is complementary to the 5 end of a sequence that is complementary to the sequence being studied, or a sequence at the 5 end of the sequence being studied. A fragment of nucleic acid (DNA or RNA) whose nucleotide sequence is complementary to that of the nucleic acid being sought, which is immobilized in the preparation (target). A sequence that is complementary to that of the target with which it specically hybridizes. A sequence that is complementary to, but of the same sense as, the target. It serves as a negative control. If a sequence is known, it is possible to make a single-strand probe, using synthetic oligonucleotides (30 nucleotides), whose sequence is complementary to that of the nucleic acid being sought (the target). A homologous copy of the target. It serves as a negative control. 401
Polymerization chamber Polynucleotide Polynucleotide kinase
Postxation Precipitation reaction
Prehybridization Pretreatment Primer
Anti-sense Sense
Probe
Anti-sense
Non-sense
Oligonucleotide
Sense
Glossary Promoter A sequence in a sense strand of DNA, situated upstream from a gene to which polymerase RNA links before the start of the transcription process. A macromolecule composed of amino acids. An enzyme that hydrolyzes proteins to form amino acids. A basic nitrogenous molecule comprising two aromatic nuclei essentially made up of the bases adenine (A) and guanine (G). It is found in nucleic acids and other cell components. A basic nitrogenous molecule with an aromatic nucleus, made up essentially of the bases cytosine (C), uracil (U), and thymine (T). It is found in nucleic acids and other cell components.
Protein Proteinase K Purine
Pyrimidine
R
Radioactivity Radioautography Recombinant DNA technology The emission of radiation by certain elements, which thereby turn into other elements. See Autoradiography. A set of techniques used in genetic engineering for the identication and isolation of a specic gene, its insertion into a vector such as a plasmid to form recombinant DNA, and, nally, the production of large quantities of the gene and its product. The rematching of a complementary nucleic acid. A process during which an exact copy of a DNA or RNA molecule is synthesized from a DNA or RNA template. A monomer of the epoxy and/or acrylic type, used for embedding tissue to make ultrathin sections. Lowicryls, LR White, Unicryl, etc. These resins are composed of acrylates or methacrylates, which are highly uid until polymerized. They have excellent penetrative properties. Epon, Araldite, Spurr, etc. These resins are composed of hydrophobic monomers whose polymerization results in very hard blocks. For example, acrylic resins. This type of resin polymerizes in the presence of a small amount of water.
Renaturation Replication
Resin
Acrylic
Epoxy
Hydrophilic
402
Glossary Hydrophobic For example, epoxy resins. This type of resin does not polymerize in the presence of water. The viscosity of acrylic resins is low (e.g., methacrylate: 0.7 centipoises at 25C), whereas that of epoxy resins is high (e.g., Araldite: 1300 to 1650 centipoises at 25C). An enzyme that breaks down RNA; see RNase. A molecule that carries genetic information. It is very similar to DNA. The sugar molecule in RNA is a ribose rather than, as in the case of DNA, a deoxyribose. A polynucleotide composed of ribonucleotides joined by phosphodiester bonds. It can take different forms: messenger RNA, transfer RNA, ribosomal RNA, or viral RNA. An RNA copy of an RNA molecule, generally obtained by in vitro transcription. Single-stranded RNA, which is synthesized from a DNA template during the transcription process. It binds to ribosomes and carries the messages required for protein synthesis. An enzyme that catalyzes the synthesis of RNA from a DNA matrix. RNA polymerase recognizes errors resulting from mismatching. Its exonucleasic activity allows it to replace incorrect nucleotides with the correct ones. RNA that is present in the nucleus and the cytoplasm. The basic component of ribosomes, which are directly involved in protein synthesis. A nuclear process during which introns are cut out of primary mRNA transcripts during its formation. A short RNA chain that transports amino acids during protein synthesis. A ribonucleic acid. In RNA, a sugar. An organelle in which proteins are synthesized and in which the messages coded in mRNA are translated. See also Ribonuclease. An enzyme that breaks down single-stranded RNA only. RNase treatment carried out after hybridization reduces background noise, and serves as a control. Experimental conditions in which all contamination by exogenous ribonuclease is eliminated, to preserve mRNA. 403
Viscosity
Ribonuclease Ribonucleic acid (RNA)
Complementary RNA (cRNA) Messenger RNA (mRNA)
Polymerase
Pre-messenger Ribosomal RNA (rRNA) Splicing
Transfer RNA (tRNA) Ribose Ribosome
RNase
RNase-free conditions
Glossary
S
Saline concentration Salinity Ionic strength. See Salinity. + The concentration of Na ions, which affects the stability of hybrids. The hybridization speed increases with the concentration of salts. See Ultramicrotomy. 0.5- to 2-m-thick sections placed on glass slides and observed at the tissue and cell levels by light microscopy. 60- to 100-nm-thick sections placed on a grid for electron microscopy. This represents the smallest quantity of target nucleic acid that can be detected in a cell or tissue, or the number of molecules that can be detected by a given label. A sequence with a primer at each end. Debrillated plasma. Serum from an animal after immunization. Serum from a nonimmunized animal of a given species. Serum from an animal before immunization. An in situ PCR/RT-PCR reaction product that shows the location of an amplied product. Detection by hybridization using a labeled probe of specic DNA fragments transferred onto a membrane. The specic activity of a probe results from its labeling, i.e., the number of isotopes or antigens incorporated, by comparison with the mass or the concentration of the probe. Total complementarity in matching between two nucleic acids. A site where the splicing of an mRNA precursor takes place. Relationship between two molecules of nucleic acid, depending on their nature. The three types of duplex that can be formed, in increasing order of stability, are DNADNA, DNARNA, RNARNA. Salts of heavy metals (e.g., uranium or lead), or tungstic acid. Heavy metal salts, which are deposited in the spaces between structures, and which thus appear light-colored against a black background.
Sections Semithin
Ultrathin Sensitivity
Sequence being sought Serum Immune Nonimmune Preimmune Signal Southern blot technique
Specic activity
Specicity Spliceosome Stability
Stain for electron microscopy Negative
404
Glossary Positive Heavy metal salts, which are deposited onto ultrathin sections so that the structures can be observed before carrying out electron microscopy. The structures appear black against a light-colored background. A process whereby an organism, a microorganism, or an enzyme is either destroyed or eliminated from an object or a solution. A protein of bacterial origin that has a very weak charge and a high afnity with four biotin molecules, and that generates little background noise. Its characteristics are similar to those of avidin, except that it has a neutral isoelectric point, and no afnity with lectins. A parameter that is used to express the efciency of hybridization and washing conditions (depending on the concentrations of salt and formamide, and the temperature). A low level of stringency favors nonspecic matchings, whereas too high a level gives rise to a specic signal of lower intensity. A substance on which an enzyme acts.
Sterilization
Streptavidin
Stringency
Substrate
T
Target Terminal deoxynucleotidyl transferase (TdT) The nucleic sequence being sought within a cell. An enzyme that is used for labeling oligonucleotide probes by elongation of the 3 end, provided that this is hydroxylated (free OH). It can polymerize NTP and dNTP. A pyrimidic base that is found in nucleosides, nucleotides, and DNA. An enzyme that catalyzes transcription. In RNA viruses it is an RNA-dependent RNA polymerase, which is used to make mRNA copies based on RNA genomes. A process in which single-stranded RNA is synthesized from a DNA template. Describes an animal or plant whose genome contains new genetic information in stable form, due to the acquisition of foreign DNA. The reading of the genetic code during protein synthesis. A nucleoside + three phosphate groups (e.g., ATP, GTP, CTP, TTP, or UTP).
Thymine Transcriptase
Transcription Transgenic
Translation Triphosphate nucleotide
405
Glossary
U
Ultramicrotome Ultramicrotomy Unicryl An apparatus for cutting resin-embedded tissue sections of variable thickness: semithin (0.5 to 2 m) or ultrathin (80 to 100 nm). A method for producing ultrathin embedded tissue sections, using an ultramicrotome. A highly hydrophilic methacrylate resin of low viscosity, giving rapid penetration of tissue. It polymerizes either at low temperature under ultraviolet radiation (25 to 35C) or with the application of heat. A pyrimidic base that is found in nucleosides, nucleotides, and RNA. A nucleotide composed of uracil.
Uracil Uridine
V
Viscosity The resistance of a uid to owing, or, in the case of a resin, to penetrating tissue. It is generally expressed in poises.
406
Index
Index
A
Acetate ammonium, 136, 356 sodium, 136, 362 uranyl, 231, 383 Acetylation, 59 Acrylic resin, 188, 190, 225, 375 Adenine, 123 AEC, 164 Agarose, 353 gel, 101, 137, 353 Alkaline phosphatase, 154 NBT/BCIP, 161, 162 endogenous, 60, 155, 162, 163, 378 revelation, 161 3-Amino-9-ethylcarbazole, see also AEC Amplication of genomic DNA, 266 Amplied product, 11 diffusion, 11, 268 AMV, 74 Antibodies, see also Immunoglobulin Antibody conjugation, 155 Anti-sense, 6, 72, 98, 125 Araldite, 188, 224 Artifact autoradiography, 117, 167 Autoclave, 351 Autoradiography, 165 artifact, 11 developer, 377 development, 166 efciency, 165 emulsion characteristics, 166 xative, 377 macro-autoradiography, 168 lm, 169 principle, 168 protocol, 169 material emulsion, 166 lm, 169 micro-autoradiography, 168 emulsion, 171 principle, 171 protocol, 172 principle, 165 protocol macroscopy, 169 light microscopy, 172 quantication, 167 resolution, 128, 167
endogenous, 96 visualization, 153, 155 Blocking agents, 222, 378 principle, 155, 159 protocol, 160 Blocking solution, 160, 221, 378 5-Bromo-4-chloro-3-indolyl phosphate, see also BCIP Buffer, acetylation, 364 blocking, 160, 221 cacodylate, 364 DNase I, 365 hybridization, 140 light microscopy, 142, post-embedding method, 228 pre-embedding method, 218 phosphate, 365 SSC, 367 Tris-EDTA, 367 Tris-HCl, 368 Buffered glycerin, 56
C
cDNA probe, 126, 138 RT, 69 Cells, 40 culture, 40, 42 xation protocol, 40, 43, 45 xation, 43 protocol monolayers, 43 pellets, 40, 45 smears, 44 suspension, 40 pellet, 40, 45 smear, 40 Chloride calcium, 356 cobalt, 133 lithium, 136, 360 magnesium, 80, 106, 360 manganese, 108 sodium, 142, 144, 363 5-Chloro-2-methoxy-benzene-diazonium chloride, see also Fast Red Chromogens, 379 alkaline phosphatase, 379 peroxidase, 380 CoCl2, see also cobalt chloride Cofactor MgCl2, 80, 106 MnCl2, 108 CoCl2, 133 Collodion lm, 226 Colloidal gold, 155, 229 Concentration Na+, 140 Contamination, 268
B
Background, 237 BCIP, 162 Biotin, 96 afnity, 96 conjugation, 97, 155
409
Index
Controls, 256 detection, 260 hybridization, 259 other techniques, 261 PCR, 259 pretreatments, 257 probe labeling, 126 reagents, 256 results, 260 reverse transcription, 258 tissue, 257 tools, 256 Counterstaining electron microscopy, 231 protocols, 231 light microscopy, 174 protocols, 174 Cover disk/cover clip protocol, 82 system, 79 cRNA, see also RNA probe Cryo-embedding, 33 Cryogenic agents, 32, 202 principles, 32 type, 32, 202 Cryo-inltration, 226 Cryopolymerization, 226 Cryoprotection, 32, 192, 200 agents, 32, 200 protocol, 32, 201, 202 Cryoprotective agents, 32 principles, 32 type, 32, 200 Cryoultramicrotomy, 192 Culture, 40 cell suspension, 40, 197 coverslide, 40 ask, 42, 198 slide, 42 Cytosine, 123 frozen tissue, 57 pepsin, 56, 57 pre-embedding method, 205 principle electron microscopy, 205 light microscopy, 54 problems, 272 pronase, 56, 57 proteinase K, 128, 163 vibratome sections principle, 205 protocol, 206 Detection, 147 autoradiographic, 165 biotin, 159 immunohistolochemical, 152 direct, 156 indirect, 156 problems, 275 Developer, 166, 169, 172, 377 Dewaxing, 53 3-Diaminobenzidine tetrachloride, see also DAB Diethylpyrocarbonate, see also DEPC Digestion of DNA, 61 Digoxigenin characteristics, 97 nucleotide conjugated, 98 Dithiothreitol, see also DTT DMSO, 33, 111 DNA, 359 double-stranded, 5, 8, 125, 130 polymerase Extapol, 108 inhibition, 107 Pfu, 107 Taq, 105 Tgo, 107 Tth, 108 repair, 264 single-stranded, 5, 9, 10, 126, 133 DNase, 61, 206, 359 dNTP, 73, 96 DTT, 80, 141, 358
D
DAB formula, 163 principle, 123 protocol, 164 Dehydration, 62 Deionized formamide, 141, 217, 228, 357 Denaturation chemical, 228, 229 principles, 216, 229 probe, 137, 143 protocol, 137, 143 Denhardts solution, 141, 217, 228 DEPC, 141, 217, 228, 355 Deproteinization, 54 chemical treatment, 58
E
Easyseal, 79, 83, 104 protocol, 83 system, 79 EDTA, 359 Efciency autoradiographic, 167 label, 135 Electron microscopy, 177 applications, 190 choice of the method, 193 methods non-embedding, 191 post-embedding, 188
410
Index
pre-embedding, 185 principle, 183 Electrophoresis, 101, 353 Embedding acrylic resin, 226 AFS, 226 cryo-embedding, 33 cryo-inltration, 226 epoxy resin, 224 oating sections acrylic resin, 226 epoxy resin, 224 Lowicryl, 226 LR White, 225, 226 parafn, 37 protocols acrylic resin, 226 epoxy resin, 224 vibratome sections acrylic resin, 226 epoxy resin, 224 Emission energy, 128, 129 Emulsion exposure, 166, 170, 174 Emulsion photographic characteristics, 171 coating, 173 detection, 174 dilution, 173 drying, 173 exposure, 166 melting, 173 storage, 173 thickness, 172 Enzyme alkaline phosphatase, 60, 154 endogenous, 60, 155, 162, 378 inhibition, 60 DNA polymerase Extapol, 108 inhibition, 107 Pfu, 107 Taq, 105 Tgo, 107 Tth, 108 DNase, 61, 206, 359 peroxidase, 154 endogenous, 60, 222 inhibition, 60 Reverse transcriptase, 74 AMV, 74 M-MLV, 75 Tth, 75 RNase, 362 Tth DNA polymerase, 75 Epi-illumination, see also Epipolarization Epipolarization, 175 Epon, 224 Ethylene diamine tetraacetic, see also EDTA Exposure time, 166, 170, 174 Extapol DNA polymerase, 108 3 Extension, 132 different stages, 133 principle, 133 protocol antigenic probe, 135 radioactive probe, 134
F
F(ab)2, 153 Fab, 153 False negative, 269 False positive, 263 Fast-Red, 162 formula, 162 FITC, see also Fluorescein Fixation, 26 criteria for choosing, 26 freezing, 31 immersion, 31 parameters, 26, 27 perfusion, 31 post-xation, 59, 207, 216 principles, 31 problems, 271 protocol, 31 cell culture, 198 cell suspension, 197 perfusion, 31 tissue, 31 whole animal, 31 Fixatives, 27, 371 chemical, 27, 221 criteria for choosing, 30 cross-linker, 27 electron microscopy, 196 formaldehyde, 28, 371 formol, 371 glutaraldehyde, 29, 196, 371 mixture, 30 osmium tetroxide, 221, 223 paraformaldehyde, 28, 372 precipitative, 27, 30 protocol, see also Fixation types, 27 Floating section, see also Vibratome section Fluorescein characteristics, 98 labeling control, 137 Formamide deionized, 141, 217, 228, 357 dimethyl, 161 Formvar lm, 226 Freezing, 31 apparatus, 33 cryogenic agents, 32 principles, 31 protocol, 32
411
Index
rapid, 33 techniques, 33 Frozen sections, 32 Frozen tissue technique, 32 pre-embedding method, 220 direct reaction, 222 indirect reaction, 223 reaction direct principles, 156 protocol, 158 streptavidin, 153 tool, 152 In vitro transcription, 127 Incorporated radioactivity, 137 Inltration, 224, 226 Ionic strength, 140, 143
G
Glutaraldehyde, 29, 196 Glycerol, 33 Guanine, 123
H
Half-life, 128, 129 Hot start, 114, 214 Hybridization, buffer, 141 cDNA, 126, 138 constituants, 141 cRNA, 140 electron microscopy, 216 frozen tissue technique, 140 oligonucleotide, 140 parameters, 138, 143 post-embedding technique, 218, 227 post-treatments, 143 pre-embedding technique, 216 principle, 216, 227 protocol, 228 principles, 123 probes, 125 protocol, 140 temperature, 138 tools, 125 Hybrids antigenic, 145 biotinylated, 145 matched, 143 mismatched, 143 radioactive, 145
K
K4M, see also Lowicryl
L
Labeling the probe, 127 PCR, 129 antigenic label, 135 3 extension, 132 principle, 129, 132 protocol, 130 radioactive label, 134 purication, 135 storage, 137 Labels, see also Biotin, Digoxigenin, Fluorescein, 35S, and 33P advantages/disadvantages, 127 antigenic, 97, 127 advantages/disadvantages, 127 characteristics, 97 controls antigenic probe, 137 radioactive probe, 137 criteria for choosing efciency, 165 resolution, 103, 128, 167 sensitivity, 165 emission energy, 128, 129 enzymatic alkaline phosphatase, 154 peroxidase, 154 principles of visualization, 161, 163 uorescent, 155 control, 137 FITC, 98 linkage, 103 particle, see also Colloidal gold position on nucleotide , 134 , 102 of label, antigenic, 97
I
IgG, see also Immunoglobulin Immunocytology, principle, 229 protocol, 230 Immunoglobulin, 153 advantages/disadvantages, 157 anti-biotin, 154 principles, 156 protocol, 158 Immunohistochemistry, 152, 220 biotin, 159 IgG, 153 indirect principles, 156 protocol, 158,
412
Index
P, 103, 134 S, 103, 134 radioactive, see also 35S, 33P advantages/disadvantages, 128 controls, 137 emission energy, 128, 129 half-life, 128, 129 position, 128 primers, 102 storage, 137 Latensication of colloidal gold, 155 Lead citrate, 231 Liquid nitrogen, 32 London resin gold, see also LR White Lowicryl, 226 LR White, 226 embedding, 226
35 33
construction, 126 criteria for choosing, 126 determination, length, 126 percentage G-C, 126 purication, 126 specic activity, 137 Osmium tetroxide, 221
P
33
M
Melting temperature, see also Tm Methyl green, 174 protocol, 174 Methylcellulose, 193, 384 MgCl2, 80, 106, 360 M-MLV, 75 MnCl2, 108 Monolayer, 43 Mounting medium, 386 aqueous, 162, 163, 165, 386 permanent, 164, 175, 387
N
Na thiosulfate, 174 NaOH, 228, 229, 363 NBT-BCIP, 161 protocol, 162 Nitroblue tetrazolium, see also NBT Nonspecic hybridization, 266 label incorporation, 264 synthesis, 265 Nucleic acids, see also DNA, RNA Nucleotides, 73, 96 antigenic, 97, 98 radioactive, 99, 103, 134
O
Observations, see also Chapter 11 bright eld, 175 dark eld, 175 epipolarization, 175 principles, 175 Oligonucleotide, see also Probes characteristics, 125
P, 99, 129 advantages/disadvantages, 103, 129 characteristics, 129 Paraformaldehyde, 29 depolymerization, 29 PCR, 87 cycle rst, 116, 215 last, 116, 215 number, 115, 215 phases, 115, 215 electron microscopy, 211 principle, 211 protocol, 219 enzymes, 104 characteristics, 107 criteria of choice, 109 Extapol, 108 inhibition, 107 Pfu, 107 Taq, 105 Tgo, 107 Tth, 108 hot start, 114, 214 principle, 91, 94 problems, 273 protocol cell suspension, 117 tissue, 112 reaction mixture direct PCR, 112 indirect PCR, 113 RT-PCR, 93 types asymmetric, 9 in situ reaction direct, 13 avantages/disadvantages, 12, 15 protocol, 112 indirect, 15 protocol, 113 nested, 9 quantitative, 9 semiquantitative, 9 symmetric, 8 Pepsin, 56, 361 Permeabilization, 53 agents, 54, 205
413
Index
principles, 53, 204 protocol, 54, 205 Peroxidase, 154, 163 endogenous, 60, 222, 379 inhibition, 60, 379 Pfu DNA polymerase,107 Photographic support, see also Emulsion or Autoradiography Poly (A), 360 Polymerase chain reaction, see also PCR Polymerization cryopolymerization, 226 epoxy resin, 225 LR White, 226 Poly (T), 10, 70 Post-embedding technique, 188 Precipitation, 135 ammonium acetate, 136, 356 ethanol, 136 sodium acetate, 136 Pre-embedding technique, 185 Preparation, 23 cells, 40 culture, 42 monolayer, 42 suspension, 45 tissue, 25 Pretreatment, see also Fixation acetylation, 59 consequence, 63 dehydration, 62 denaturation, 137, 229 deproteinisation, 54 dewaxing, 53 permeabilization, 53 post-xation, 59, 207 pre-embedding technique, deproteinization, 205 permeabilisation, 202 principles, 185 principle, 51 slides, 352 Primers, 6, 99 anti-sense, 6, 98 labeled, 102 5 extension, 103 antigenic, 104 radioactive, 102 poly (T), 10, 70 random, 10, 71 sense, 6, 98 specic, 10, 72 characteristics, 72, 100 concentration, 101 hybridization temperature, 73, 100 position, 73, 100 storage, 101 validation, 101 Probes, 125 anti-sense, 125 characteristics, 125 controls, 126 denition, 125 denaturation, 137 DNA double-stranded, 126 single-stranded, 126 labeling techniques, 127 length, 126 oligonucleotide characteristics, 125 construction, 126 criteria for choosing, 126 length, 126 percentage G-C, 126 purication, 126 specic activity, 137 post-treatment, 143 RNA, see also In vitro transcription sense, 125 storage, 137 type, 126 utilization, 137 washes, 143 Problems, see Chapter 9 Pronase, 56, 361 Proteinase, 360 Proteinase K, 55, 360 concentration, 55 parameters, 55 protocol, 56 use, 55 Purication, 135 protocol, 136
R
Resin acrylic, 225 Lowicryl, 225 LR White, 225 Resolution autoradiography, 167 Revelation, 147, 377 autoradiography, 165 immunohistochemistry, 152 principle, 150 Reverse transcriptase, 74 AMV, 74 criteria of choice, 76 M-MLV, 75 Tth, 75 Reverse Transcription, 9, 65 electron microscopy, 207 pre-embedding method, 207 principle, 208 protocol, 209 principle, 69 problems, 272
414
Index
protocol, 80 cell suspension, 81, 85 tissue section, 81, 86 tools, 70 primer, 70 dNTP, 73 enzyme, 74 materials, 77 types asymmetrical, 9 differential display, 9 symmetrical, 9 RNA, 361 RNase A, 362 RNase free conditions, 349 RNasin, 80 RT, see also Reverse transcription Signal/background ratio, 237 Slides preparation, 352 Sodium hydroxide, see also NaOH Specic activity, 137 Specicity, 243 SSC, 367 Stabilization of structures, see also Fixation Staining electron microscopy, 231, 383 lead citrate, 231, 384 uranyl acetate, 231, 383 light microscopy, 380 cresyl violet, 380 eosin, 381 hematoxylin, 381 methyl green, 174, 382 nuclear red, 382 toluidine blue, 174, 382 Sterilization, 351 Storage primer, 111 probe, 137 sample, 34 solutions, see Appendices thin section, 62 ultrathin section, 232 vibratome section, 202 Streptavidin, 153, 155 Streptavidin/biotin complex, 153, 155 Sucrose, 32
S
S, 128 advantages/disadvantages, 128 characteristics, 128 Safe-light, 172 Sample electron microscopy, 195 cell, 195 tissue, 196 light microscopy, 21 cell, 40 tissue, 25 preparation, see also Fixation Sarcosyl, 362 Sealing system, 79 cover disk/cover clip, 79 easyseal, 79 protocol, 110 cover disk/cover clip, 82 easyseal, 83 Sections frozen, 34 parafn, 38 staining, electron microscopy, 231 light microscopy, 174 storage thin, 62 ultrathin, 232 vibratome, 202 ultrathin, 226 vibratome, 199 parameters, 199 storage, 202 Sense, 6, 218 Sensitivity, 243 Sequence, 5 anti-sense, 6 sense, 6 target, 5, 11
35
T
Taq DNA polymerase, 105 Target sequence denition, 5, 11 destruction, 270 TdT, see also Terminal deoxytransferase Techniques for freezing tissue, 32 Techniques for labeling, see also 5 Extension, 3 Extension, and Symmetric PCR Terminal deoxinucleotide transferase, 132 Tgo DNA polymerase, 107 Thermocycler materials, 77, 78, 109 programming, 116 Thymidine, 123 Tm, 138 Toluidine blue, 174 Tris, 364 Triton X-100, 364 Tw, see also Wash temperature Typical protocols, see Chapter 10
U
Ultramicrotome, 226 Ultrathin section, 226, 231, 232 Uracile, 123
415
Index
V
Vibratome equipment, 199 section embedding epoxy resin, 224 LR White, 226 parameters, 199 protocol, 201 storage, 202 Visualization alkaline phosphatase, 161 autoradiography, 165 macro-autoradiography, 169 micro-autoradiography, 172 biotin, 159 immunohistochemistry, 152 reaction direct, 158 indirect, 158
peroxidase, 163 pre-embedding technique ultrathin section, 230 vibratome section, 222 Vitamin H, see also Biotin
W
Washes light microscopy, 143 pre-embedding method, ultrathin section, 229 vibratome section, 219 problems, 275 temperature, 144 Water DEPC, 355 sterile, 355
416

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PCR/RT-PCR in situ

LIGHT and ELECTRON MICROSCOPY

Library of Congress Cataloging-in-Publication Data

Visit the CRC Press Web site at www.crcpress.com

Polymerase Chain Reaction (PCR)

1.1 POLYMERASE CHAIN REACTION (PCR)

1.1.2 The Target Sequence

2 copies, where n = the number of cycles.

1.1 1.1.3.2 Cycle

Polymerase Chain Reaction (PCR)

The PCR reaction comprises a three-stage cycle: Denaturation Hybridization Extension

Gloves must be worn.

1.1.5 The Main Types of PCR

1.2 Asymmetrical PCR

Reverse Transcription (RT)

1.2 REVERSE TRANSCRIPTION (RT)

See Chapter 4. See Section 4.3.1. See Section 4.3.3.

Stabilize a solution of different types of RNA by turning them into cDNA

The hybridization of the primer

1.2.2 The Main Types of RT

1.3 IN SITU PCR/RT-PCR

The tissue structure

The cell structure

And facilitating the accessibility of The sequence of interest

The amplied products

While avoiding The diffusion of the amplied product

General Principles Permeabilized cytoplasmic and nuclear membranes.

Accessible DNA or RNA target sequences Figure 1.5 A pretreated cell.

Limited efciency of the amplication

Diffusion of the amplied products

The partial destruction of morphology

The necessity for specic equipment

1.3.2 In Situ Amplication Methods

Direct In Situ PCR/RT-PCR

1.4 DIRECT IN SITU PCR/RT-PCR

z The DNA target sequences amplied and directly labeled

y Direct marking of the amplied product

Figure 1.10 Amplication of retrotranscribed cDNA. 14

Indirect In Situ PCR/RT-PCR

See Section 9.3. See Section 9.4.

1.5 INDIRECT IN SITU PCR/RT-PCR

z Amplied DNA sequences

{ Hybridization with a pair of labeled primers (see Chapter 5)

Indirect In Situ PCR/RT-PCR

} Immunocytological detection (antigenic label) See Section 7.2.

~ Autoradiographic detection (radioactive label) See Section 7.3.

General Principles z Sequences of the amplied, unlabeled cDNA of interest

{ Hybridization with a pair of labeled primers (see Chapter 5)

} Autoradiographic detection (radioactive label) See Section 7.3.

~ Immunocytological detection (antigenic label) See Section 7.2.

Figure 1.14 Indirect in situ RT-PCR.

The estimated number of copies per cell

2.1 TISSUE SAMPLES

2.1.1 Sampling Conditions

Fixation, followed by: Freezing Parafn or Paraplast embedding

Freezing, without xation, followed by: Fixation on slides

2.1.2 Diagram of the Different Steps

The satisfactory xation of tissue depends on a number of factors:

Precipitating xatives Fixative mixtures

A high degree of conservation of proteins, which satises morphological requirements

Figure 2.3 Formula of glutaraldehyde.

Working solution (see Appendix B4.3)