HISTOLOGY

LECTURE # 12

INTRODUCTION TO TISSUE FIXATION

Rationale: Fixation is the most essential part in histology. Here is where everything starts. A well fixed tissue is the key for a good slide and therefore a good interpretation for diagnosis. Here we will learn the several types of fixation available their advantages and disadvantages.

Objective:
Once completed this lecture, the student should be able to: a) b) c) d) Describe the various fixatives and their uses. Learn the difference between autolysis and putrefaction Differentiate the fixatives that could impact the final results. Learn the chemicals and reagents used in each fixative.

TISSUE FIXATIVE
Introduction
Fixation - is the most important step through the process of histology. The purpose of the fixative is to stabilize the protein in the tissue. Once the tissue is removed from the body it will go through a process of self-destruction. This process is known as Autolysis which starts soon after the cell death creating an enzyme attack, which in place causes the breakdown of protein and eventual liquefaction of the cell.
Autolysis is more severe in tissues which are rich in enzymes, such as the liver, brain and kidney, and is less rapid in tissues such as elastic fibers and collagen. By light microscopy, autolyzed tissue presents a `washed-out' appearance with swelling of cytoplasm, eventually converting to a granular, homogeneous mass which fails to take up stains. If tissue is left without any preservation, then a bacterial attack will occur, this process is known as Putrefaction.

The objective of fixation is to preserve cells and tissue constituents in as close a life-like state as possible and to allow them to undergo further preparative procedures without change. Fixation arrests autolysis and bacterial decomposition and stabilizes the cellular and tissue constituents so that they withstand the subsequent stages of tissue processing. Fixation should also provide for the preservation of tissue substances and proteins, therefore, it is the first step and the foundation in a sequence of events that culminates in the final examination of a tissue section.

FUNCTION OF FIXATIVES
A. Help Maintain a proper relationship between cells and extracellular substances: Connective Tissue Fibers: Collagen, Reticulin, Elastin Amorphous ground substances

B. Brings out differences in refractive indexes and increases the visibility or contrast between different tissue elements. R.I. = Velocity of light in Air/Velocity of light in a liquid or solid medium C. Render cell constituents insoluble, with tissue proteins serving as the primary target for stabilization.

ACTION OF FIXATIVES
A. Methods of Stabilizing Proteins 1. 2. 3. Heat (Physical method) this method is becoming more used in the histology laboratories with the introduction of microwave fixation. Desiccation (Physical method) Rarely used in histology. Touch preparation s for Giemsa stains are probably the most common used for this method. The use of one or more chemical reagents (Chemical method) this is considered the primary method of fixation. These chemical reagents may be classified as additive and non-additive or coagulant and non-coagulant. Additive they chemically link or bind to the tissue and change it. Non-additive include organic compounds such as acetone and alcohols, which act on the tissue without chemically combining with the tissue. Ex: Methyl or Ethyl Alcohols B. Coagulant & Non-Coagulant 1. Coagulation will allow the solutions to penetrate into the interior of the tissue very easily. Non-coagulant - they act by creating a gel barrier that makes solutions more difficult to penetrate to the interior of the tissue.

2.

To further understand this concept of coagulant and non-coagulant, take a look to the following examples:

(A) Coagulant

(B) Non-Coagulant

Imagine that we have a piece of tissue in the middle of both examples. Which will you think that the solutions will penetrate easily to the tissue? Answer: (A) Coagulant: these types of fixative will render the tissue so that it will accept subsequent solution very easily. If we look at (B) Non-coagulant, theses types of fixative will make it difficult for subsequent solutions to penetrate. Coagulant Fixatives Reagents includes: Alcohol Zinc salts Mercuric chloride Chromium trioxide Picric Acid

Non-Coagulant Fixatives Reagents includes: Formaldehyde Gluteraldehyde Osmium Tetroxide Potassium Dichromate Acetic Acid

FACTORS AFFECTING FIXATION

Temperature
The factor influenced by temperature is the morphology of the tissue. Normally the fixation of specimens for standard histology is carried out at room temperature for convenience. For electron microscopy and some histochemical procedures, the temperature for fixation is usually 0-4C. The use of chemical reactions at higher temperatures to penetrate tissue, including those involved in the fixation process will increase the rate of penetration of the fixative to the specimen, however, an increase in temperature will also increase the rate of autolysis and diffusion of cellular components.

Size
The penetration of fixatives into tissue is a relatively slow process and tissue blocks should either be small or thin, in order to obtain satisfactory fixation. Large specimens should be opened and washed of contents or sliced thinly before placement in fixative. For routine processing the recommended thickness of the specimens should be no more than 3mm

thick (not thicker than a nickel). For shorter processing schedules the tissue should be of thinner sections, Ex: Kidney, liver, heart biopsies)

Volume Ratio
The recommended ratio of the tissue volume to the fixative volume is at least 15 to 20 times greater than the tissue volume. If a specimen receives more than the recommended volume the effect is none, we have plenty of fixative to the tissue volume. However, if the volume of the fixative is less than the volume of the tissue, we can see some problems such as under fixed tissue (poor fixation).

S S S

S F F S

Fixative S Tissue S

S S SS S FFF F F S S F F S S S S SS

Time
It is common practice to fix 2 mm thick tissue blocks in buffered formalin for 4-8 hours, possibly followed by a period in formol sublimate. Large specimens and viscera are cut into 5 mm slices or viscera are emptied and pinned out on a board, before fixing overnight in buffered formalin. This allows easier handling, examination and dissection, particularly for the sampling of lymph nodes. In the case of electron microscopy, diced tissues are fixed for 3 hours in glutaraldehyde before placing in a holding buffer such as sodium cacodylate. There is evidence that prolonged fixation in aldehydes can cause shrinkage and hardening of tissue and severe inhibition of enzyme activity. Prolonged fixation with oxidizing fixatives can degrade tissues by oxidative cleavage of proteins and loss of peptides. The tissue should be placed in fixative immediately after surgery, as well as autopsies should be performed immediately after death. The more time it elapses between interruption of the blood supply and fixation, the more post-mortem changes will be observed under the microscope.

Small intestine well preserved

Autolyzed Small intestine Carson Book, Page 5, Image 1-2 Notice how is missing the epithelium

Choice of Fixative
The method of fixation should be selected immediately once the specimen is presented. The fixative should be selected with caution avoiding fixatives that may affect future histochemical studies. For example tissue that will be used for Immunofluorescence studies should be preserved in a transport media. If future studies include a diagnostic for Gout (Gophus Typhus), a fixative containing water will not work since the water will dissolve the Urate crystals which are essential in the study, so the fixative of choice is absolute alcohol. Enzyme studies are also very peculiar when it comes to fixatives, frozen sections are preferred for these methods. Electron Microscopy the choice is Gluteraldehyde. Post-fixation which is also considered a mordanting process will help with the enhancement of the staining by allowing a better penetration of solution to the tissue structures to be observed. Example: Masson Trichrome.

Solution Reagents

Zenkers Mercuric Chloride Potassium Dichromate Acetic Acid

Hellys Mercuric Chloride Potassium Dichromate

B-5 Mercuric Chloride Sodium Acetate

Bouins Picric Acid Formaldehyde 37 40% Acetic Acid Yellow

Formaldehyde Formaldehyde 37 40% 37 40% Orange Transparent

Color

Orange

Tissue

Bone Marrow Bone Marrow Biopsies Aspirates

Bone Marrow Cores Tumors

GI Biopsies

Solution Reagents

Hollandes Copper Acetate Picric Acid

Orths Sodium Sulfate Potassium Dichromate Formaldehyde 37 40%

Zambonis Picric Acid Sodium Phosphate, Mono Sodium Phosphate, Dibasic Formaldehyde 37 40%

Carnoys Absolute Alcohol Chloroform

Acetic Acid

Acetic Acid

Formaldehyde 37 40% Color Green Orange

Yellow

Clear

Tissue

Small Decals Bones

Adrenal Medulla

EM Fixative

Lyse Red Blood Cells

Solution

10% Formalin

10% Formol Saline

Neutral Buffered Formalin

Formalin Ammonium Bromide

Reagents

Formaldehyde Formaldehyde Formaldehyde Formaldehyde 37 40% 37 40% 37 40% 37 40% Distilled Water Sodium Chloride Sodium Phosphate, Mono Sodium Phosphate, Dibasic Distilled Water Distilled Water Clear Prevent Pigments Gluteraldehyd e Cocodylate Buffer Gluteraldehyd e Stock Distilled Water Clear Clear Distilled Water Clear Brain Tissue Ammonium Bromide

Color Tissue

Clear Routine

Clear Routine

Solution Reagents

10% Formal Alcohol Formaldehyde 37 40% 95% Alcohol

Flemmings 2% Osmium Tetroxide 1% Chromium Hydroxide Acetic Acid

Schaudinns Mercuric Chloride in Water Absolute Alcohol

Color Tissue

Clear

Clear

EM Specimen EM Specimen EM Specimen EM Specimen

Penetration
Fixatives penetrate the tissue at different rates. These rates can be affected by heat. The tissue is fixed starting at the periphery of the tissue and working inward toward the center of the tissue.

Tissue Storage
Storing wet tissue is very important because often the tissue is needed for further studies. If the tissue has not been fixed and stored properly further studies are impossible. Tissue fixed in Neutral buffered Formalin are usually safe to use for further studies since they can remain in this fixative indefinitely, but this is not true for other fixatives. Non fix tissue may remain in 70% methyl alcohol.

pH
The hydrogen ion concentration varies between fixatives, but in general, the pH should be kept in the physiological range, between pH 4-9. If formalin is allowed to fall to a lower pH this can produce formalin pigments. Even though in routine histology the pH is not usually critical, in electron microscopy it is very important. The pH for the ultrastructural preservation of great specimen the fixative should be buffered between 7.2 to 7.4.

Osmolality
The addition of a buffer to the fixative solution may alter the osmotic pressure exerted by the solution. Hypertonic solutions give rise to cell shrinkage whereas isotonic and hypotonic fixatives result in cell swelling and poor fixation. With electron microscopy, the best results are obtained using slightly hypertonic solutions (isotonic solutions being 340 mOsm) adjusted using sucrose.

REACTIONS OF THE CELL WITH FIXATIVES

Glycogen A variety of glycogens occurs naturally and show different degrees of polymerization. The less highly polymerized forms are not well fixed by routine fixatives and diffuse into the fixing fluid. This occurs in cases of glycogen storage disease where glycogen is predominantly of the lighter type. In contrast, the larger molecules of more highly polymerized glycogens are retained with a wide variety of fixatives as well as alcoholcontaining reagents. The retention of glycogen is thought to be the result of trapping in a matrix or mesh of fixed protein, or due to its covalent binding to protein which renders it insoluble in water. However, there appears to be stronger support for the concept that removal, by dehydration, of bound water molecules from normal forms of tissue glycogen decreases solubility, amounting to denaturation.

Lipids With standard methods of fixation, lipids are largely lost from tissues during processing and only two reagents fix lipids in the true sense of rendering them insoluble. These are osmium tetroxide and chromic acid, both of which alter the chemical reactivity of the lipid considerably. While several fixatives will preserve lipids, they generally do not alter their solubility in the lipid solvents used in tissue processing. Baker's fixative, designed for the preservation of phospholipids, uses formalin together with calcium and cadmium chlorides (the last, being expensive, has subsequently been replaced by cobalt nitrate). While phospholipids are preserved, they are not prevented from diffusing into the fixing fluid and are still removable by fat solvents. Lipids can be demonstrated in cryostat sections fixed with reagents containing mercuric chloride and potassium dichromate such as Elftman's fluid, with fixation for unsaturated lipids completed over 3 days at room temperature. Various additives have been mixed with glutaraldehyde in order to demonstrate lipids in electron microscopy. Digitonin added to glutaraldehyde preserves cholesterol and Malachite Green included with glutaraldehyde or Karnovsky's fixative retains various lipids such as phospholipids, fatty acids, glycolipids and choline plasmalogen. Imidazole introduced into the post osmication of glutaraldehyde-fixed tissue demonstrates unsaturated fatty acids and phospholipids as electron-dense deposits. Proteins The fixation of tissue proteins by aldehydes is largely through production of cross-linkages between various reactive groups in proteins. Most fixatives preserve proteins adequately in 1 to 2 days. Glutaraldehyde fixes proteins very rapidly whereas formaldehyde reacts reversibly over the first 24 hours. Osmium tetroxide reacts with proteins by producing cross-links and protein gels. Prolonged exposure to osmium tetroxide causes the breakdown of proteins. Mucosubstances Among the mucosubstances are the single component polysaccharides such as glucose, starch and cellulose which are referred to as homoglycans whereas those with two or more monosaccharide components are the heteroglycans. The latter are composed of the glycosaminoglycans such as keratosulphate and sialoglycans, and the glycosaminoglucoronoglycans comprising hyaluronic acid, chondroitin sulphates and heparin. Protein-polysaccharide complexes are known as proteoglycans. The loss of mucosubstances from tissue during fixation is well recognized and many fixatives have been suggested to prevent this. Formalin has always been an essential component of whatever fixative used to ensure the preservation of proteoglycans, however, an appreciable proportion of tissue hetero- and proteoglycans remains soluble unless subject to further precipitation in 70-80% ethanol (for 3-6 days) before clearing and embedding in paraffin. Nucleic acids and nucleoproteins The nucleic acids exist in many different states of polymerization and any method of fixation induces changes in their physical state. Formalin is not a particularly good fixative for nucleic acids and nucleoproteins as it blocks a large number of reactive groups reducing their subsequent staining by both basic and acid dyes. This can be improved by adding mercury or chromium salts. Precipitant fixatives like alcohol, acetic acid, and Carnoy's fluid are preferable since these agents precipitate nuclear proteins and at the same time progressively break the bonds between nucleic acids and proteins, thereby increasing the number of acid groups available for staining. However, prolonged fixation in acid fixatives such as Carnoy's reagent profoundly alters nuclear proteins and extracts RNA and DNA.

Simple Aqueous Fixatives or Fixative Ingredients

FORMALDEHYDE Formaldehyde, as 4% buffered formaldehyde (10% buffered formalin), is the most widely employed universal fixative particularly for routine paraffin embedded sections. It is a gas with a very pungent odor, soluble in water to a maximum extent of 40% by weight and is sold as such under the name of formaldehyde (40%) or formalin (a colorless liquid). Formaldehyde is also obtainable in a stable solid form composed of high molecular weight polymers known as Para formaldehyde. Heated Para formaldehyde generates pure gaseous formaldehyde which, when dissolved in water, reverts mostly to the monomer form. Aqueous formaldehyde exists principally in the form of its monohydrate, Methylene glycol, CH2(OH)2, and as low molecular weight polymeric hydrates or polyoxymethylene glycols. It has been suggested that the hydrated form, Methylene glycol, is the reactive component of formaldehyde but this has been disputed. Four per cent formaldehyde or 10% buffered formalin is commonly prepared by adding 100 ml of 40% formaldehyde to 900 ml distilled water with 4 g sodium phosphatase, monobasic and 6.5 g sodium phosphate, dibasic (anhydrous). To be effective, the specimen should be completely submerged in five to twenty times its volume of fixative. Ten per cent buffered neutral formalin preserves a wide range of tissues and has the advantage of being a forgiving fixative. It requires a relatively short fixation time but can also be used for long-term storage as it produces no deleterious effects on tissue morphology with nuclear and cytoplasmic detail being adequately preserved. Details of the fixing action of formaldehyde and other aldehydes are not known although the general principles are understood. It is thought that the aldehydes form cross-links between proteins, creating a gel, thus retaining cellular constituents in their in vivo relationships to each other. Soluble proteins are fixed to structural proteins and rendered insoluble, giving some mechanical strength to the entire structure which enables it to withstand subsequent processing. With the aldehydes, cross-links are formed between protein molecules, the reaction being mostly with basic amino acid lysine, although other groups such as imino, amido, peptide, guanidyl, hydroxyl, carboxyl, SH and aromatic rings may also be involved. Only those lysine residues which are on the exterior of the protein molecule react, these usually accounting for 40-60% of the total lysyl residues. Although the extent of denaturation produced by fixation this does not matter greatly in routine tissue pathology, it is of particular importance in the detection of antigens both by immunofluorescence and immunoenzyme techniques as well as in high resolution electron microscopy. Similarly, the shapes of large molecules must not be changed if they are to be recognized by biochemical analysis. Glutaraldehyde causes a loss of up to 30% of the alpha helix structure of protein, depending on the type of protein. Fixation with osmium tetroxide or post osmication of glutaraldehyde-fixed material causes the complete denaturation of protein. Formalin does not precipitate proteins and only slightly precipitates other components of the cell. It does not harden or render albumin insoluble but subsequent hardening by alcohols is prevented. Formalin neither preserves nor destroys adipose tissue and is a good fixative for complex lipids but has no effect on neutral fats. Although not the fixative of choice for carbohydrates it preserves proteins so that they hold glycogen which is otherwise readily leeched from the cell. Formaldehyde solution is nearly always acid. It certainly becomes acid on storage as formalin oxidizes to formic acid, reducing its preserving capabilities such that neutralization of the solution is a requirement. In addition, formaldehyde solution produces acid formalin haematin pigment which can be seen in sites containing blood. If calcium carbonate is used for neutralizing formalin the resultant solution does not retain its neutral pH and calcium carbonate itself can deposit in tissues, leaving areas of `pseudocalcification'. Phosphate

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buffers such as sodium phosphate monobasic and sodium phosphate dibasic are effective for the neutralization of formalin and the pH of the solution produced is stable for many months. Formalin should not be used with chromates because it readily oxidizes to formic acid. A concentrated solution of formalin sometimes becomes turbid on storage through the production of Para formaldehyde which decreases the strength of the solution, but can still be used as a fixative following filtration. Formalin favors the staining of acidic structures (nuclei) with basic dyes and diminishes the effect of acid dyes on basic structures (cytoplasm). Formaldehyde is an immediate irritant to the eyes, upper respiratory tract and the skin, and safety precautions should include proper ventilation and exhaust, limited or restricted exposure periods and thorough washing if spilt on tissue surfaces such as the skin. GLUTARALDEHYDE Like formaldehyde which acts through the formation of cross-links between protein endgroups, glutaraldehyde has also been used extensively as an agent for protein-protein linkage and hence for fixation. An aqueous solution of glutaraldehyde (glutaric dialdehyde) is a complex mixture at room temperature, consisting of approximately 4% free aldehyde, 16% monohydrate, 9% dihydrate and 70% hemiacetal. Free glutaraldehyde may form polymers, or a monohydrate and a dihydrate, which may cyclize to give a hemiacetal which in turn may also polymerize. Some favor the polymer as the reactive species while others suggest that pure, monomer, glutaraldehyde is the best fixative and much less inhibitory to enzymes than is the mixed monomer-polymeric product. The success of glutaraldehyde as a cross-linking agent may also depend on the large range of different molecules present simultaneously in the fixation solution. When glutaraldehyde solutions are kept for long periods at ambient temperatures, there is a tendency for precipitates to form and for aldehyde levels to fall so that some method of purification may be required. Glutaraldehyde may be purified to the monomer form by removing oligomers, polymers and other impurities through simple shaking with barium carbonate, vacuum distillation or treatment with activated charcoal and chromatography on Sephadex G-10 has produced equally good results. Vacuum distillation after prior treatment of commercial glutaraldehyde solutions with sodium chloride and ethanol has become the most widely used technique for purification. There are many variations in the preparation of this fixative, including the percentage of glutaraldehyde, additives, and buffers. Because of its low penetration, only small blocks of tissues (1-2 mm3) fix well at temperatures of 1-4C. The fixed tissue specimen can be stored in buffer solution for many months. The slow penetration, the requirement for cold temperature and the need for a storage medium, have greatly limited the use of glutaraldehyde in histology. It is, however, the most widely used fixative for standard electron microscopy. Other uses for glutaraldehyde all of which really its cross-linking properties include the preparation of tissue xenografts, particularly cardiac valves, chemical sterilisation and disinfection. Glutaraldehyde has an inhibitory effect on catalase allowing the selective demonstration of the peroxidase activity of peroxisomes. OSMIUM TETROXIDE The most commonly used metallic ion in fixation is osmium tetroxide which was initially a tissue fixative used in cytology, but poor penetration limited its application in light microscopy. It is now largely employed as a secondary fixative in electron microscopy. Osmium tetroxide is known to form cross-links with proteins as reflected in the rapid increase in viscosity of a protein solution when they react together, however, there is very little additional information as to its the mechanism of action. There is some general

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agreement that osmium tetroxide reacts with unsaturated lipids as it is reduced with the formation of black compounds containing hexavalent osmium. Various hypotheses of lipid stabilization have been postulated and these include the oxidation of double bonds between adjacent carbon atoms to form monoesters and diesters, the binding of lipid to protein and the conversion of unsaturated fatty acids to stable glycol osmates. More recent studies show that the reaction of osmium tetroxide is largely with lipid rather than protein. Osmium tetroxide is used for preservation of fine structures in electron microscopy and is effective for small (2-3 mm3) specimens. While vapors of this fixative will preserve blood and tissue smears, its low and uneven penetration limits its application in routine light microscopy and osmium tetroxide fixed tissues often crumble if embedded in paraffin. Osmium tetroxide also interferes with many staining procedures. CHROMIC ACID Chromic acid (chromium trioxide) is a strong oxidizer that is used with other ingredients. It has no effect on fats, penetrates slowly and leaves tissues in a state where shrinkage may occur during subsequent processing. Chromium salts form complexes with water which combine with reactive groups of adjacent protein chains to bring about a cross-linking effect similar to that of formalin. The reaction of potassium dichromate with adrenal medullary catecholamines results in the production of black or brown water-insoluble precipitates. The dichromate-oxidation product is not only visible grossly but also in the tissue section and is still regarded as a rapid means of identifying tissues with aromatic amines such as adrenal medullary tumors. Potassium dichromate is never used alone and, if employed other than for the demonstration of amines, should be washed thoroughly to remove the oxide that forms as it cannot be removed later in processing. Other heavy metals such as palladium chloride and uranium may result in some degree of tissue fixation but have no practical application in histopathology. ACETIC ACID Acetic acid is never used alone but is often combined with other fixatives that cause shrinkage such as ethanol and methanol. Acetic acid penetrates thoroughly and rapidly but lyses red blood cells. PICRIC ACID Picric acid, when used in combination with other ingredients, leaves tissue soft and penetrates well, precipitating all proteins. It will continue to react with the tissue structures and cause a loss of basophilia unless the specimen is thoroughly washed following fixation. MERCURIC CHLORIDE Mercuric chloride (corrosive sublimate, bichloride of mercury) and other salts of mercury were common histological fixatives in the past. These penetrate rapidly and precipitate all proteins, reacting with a number of amino acid residues including thiol, amino, imidazole, phosphate and hydroxyl groups. The production of hydrogen ions makes the fixative solution more acidic and mercuric crystals deposited in the tissue need to be removed before staining. Mercuric chloride is contained in Zenker's, Helly's, Ridley's and B5 solutions. It should also be noted that mercuric salts are highly toxic and must not be disposed into sewerage systems. One method of disposal is to precipitate the mercuric chloride with thioacetamide. For example, mixing 1 liter of Zenker's solution with 20 ml of 13% thioacetamide solution in a tightly capped container results in a precipitate of mercuric sulphide which can be filtered out and disposed of safely. ACETONE Acetone is a clear, colorless, inflammable liquid which is miscible with water, ethanol and most organic solvents. It has been used as a dehydrating agent in tissue processing and is more volatile than alcohols and other dehydrants. It has a rapid action but causes brittleness in tissue if exposure is prolonged and because it is volatile and inflammable, acetone is not used in automated processing schedules. However, it has a greater solvent action on lipids and is rapidly removed by most clearing agents, making it very useful in manual processing procedures.

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More recently, acetone has been employed as a fixative in the acetone-methylbenzoatexylene (AMEX) technique.12 This requires overnight fixation of tissues in acetone at -20C then clearing in methylbenzoate and xylene before paraffin embedding. The product is claimed to show better histologic preservation than is possible to obtain in frozen sections, yet retaining reactivity for labile lymphocyte membrane antigens. TRANSPORT SOLUTIONS Michel Transport Medium It is important to maintain the pH in between 7.0 to 7.2, because a lower pH can cause variable results. This transport medium is used for specimen such as kidney biopsies that need to be frozen for further immunohistochemical studies. REMOVAL OF FIXATION PIGMENTS Formalin Pigments these can be removed by immersing the sections in saturated absolute alcohol with picric acid for 10 minutes to three hours. Then wash sections well with water. Also, a solution of 70% alcohol containing 3 mL of ammonium hydroxide for 30 minutes to 3 hours. After treatment wash sections well in 1% acetic acid. Mercury Pigments these can be removed by immersing the sections in Gram or Lugols Iodine for 10 minutes, and then place the section in a 5% solution of Sodium thiosulfate for 3 minutes. Wash slides well in running water for 10 minutes. Melanin Pigments this is removed by placing the section in a solution of Potassium permanganate for approximately 20 minutes, followed by a solution of Oxalic acid to remove the excess of Potassium permanganate. Fixatives: a summary It is clear that there is no universal fixative which will serve all requirements. Each fixative has specific properties and disadvantages and their many different effects emphasize the necessity for careful consideration and selection of the appropriate fixing reagent when studies of specific cellular substances are planned. Ten per cent buffered formalin and 2.5% Gluteraldehyde are currently the most widely used fixatives for routine light microscopy and ultrastructural studies, but they too, have inherent disadvantages which the user should be well conversant with. Increasing interest in tissue and cell constituents including cellular proteins detectable by immunohistochemical techniques imposes additional requirements for the preservation of such substances.

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