Visualization

Techniques Used in
Histology
Alvin Rey Flores, RMT, MT(ASCPi), MPH

Visualization Techniques
In elucida<ng func<onal aspects of the cells, <ssues, and
organs being studied
Most commonly used techniques are rou<ne staining;
histochemistry (and cytochemistry),
immunocytochemistry, autoradiography, column
chromatography, and gene analysis
Use chemical reac<ons, enzyma<c processes, and
physicochemical processes that not only stain the <ssue
but also permit the localiza<on of extracellular and
intracellular molecules of interest

Routine Staining
ROUTINE STAINING USING H and E
H and E is a combina<on of a Nuclear and Cytoplasmic Stain
Commonly Employed in Histology
Hematoxylin and Eosin

Autoradiography
Localiza<on of Newly Synthesized Macromolecules (DNA,
RNA, polysaccharides, glycoproteins) in cells or <ssues
Use of radioac<ve labels which emit small amounts of
radia<on
Radiolabeled <ssue are coated with Silver Bromide which
act as microdetectors of radia<on
Can be applied in LM and EM

Cell and Tissue Culture

Used in the isola<on and propaga<on of viruses and other
bacteria that are dicult to cul<vate in ar<cial media
Useful in Isola<ng the eects of single molecules on specic
cell types.
Direct Observa<on can be done using the a phase contrast
microscope
Cells are grown in complex solu<ons with known composi<on

 Primary Cell Cultures derived from parent <ssue

ex. Human Embryonic Kidney, Rabbit Kidney
Immortal Cell Lines
- ex. HeLa cell line, Hep-2;

Immunohistochemistry
Identification of specific or selective tissue or
cellular antigens
Used for detection of organisms in cytologic
preparations (eg. Fluids, sputum samples, FNA)
Makes use of antigen-antibody reactions
Labels are used to iden<fy <ssues or cells of interests
- Enzyme Labels Include Horseradish Peroxidase
- Chromogens such as FITC (uorescein
isothiocyanate)

Enzyme Histochemistry
Histochemical Staining
Study of the <ssue cons<tuents through their chemical
reac<ons
Microscopic Localiza<on of Specic Tissue Substances
The process whereby various cons<tuents of <ssues

Enzyme Histochemistry
- uses enzymes ( such as acid phosphatase,
dehydrogenases, and peroxidases) in specic cell
organelles to localize those components specically
- Enzymes Detected: Phosphatases, Dehydrogenases,
peroxidases

Hybridization Techniques
(Molecular Techniques)
Hybridiza<on binding between two single strands of
nucleic acids that recognize together if they are
complementary.
- More Similar; More Complementary

Hybridization Techniques
(Molecular Techniques)
IN-SITU Hybridiza<on
- Hybridiza<on on site
- Solu<ons of nucleic acids are applied DIRECTLY on the
<ssue
USES: determina<on of a specic DNA sequence
determina<on of specic mRNA
determina<on of the localiza<on of a gene

Other Molecular Techniques

GENE SPLICING
Genes from one organism can be spliced into another using a
technique called gene splicing.

Electrophoresis
Electrophoresis is a method of separa<ng of protein
fragments of dierent lengths. The protein samples are
placed in <ny "wells" at one end of an agarose gel. An
electric current is then passed over the gel, separa<ng
the fragments.

Immunoblot Techniques
Types of Blo`ng Techniques

A. Western Blot
B. Northern Blot
C. Southern Blot

Polymerase Chain Reaction

Gene Amplica<on
3 Main Steps
1. Denatura<on
2. Annealing
3. Elonga<on