FixativesAldehydes and other cross-linking fixativesFORMALDEHYDEFormaldehyde, as 4% buffered formaldehyde (10% buffered formalin), is the most widely employed universal fixative particularly for routine paraffin embedded sections. Formaldehyde which acts through the formation of cross-links between protein end-groups.

GLUTARALDEHYDEIt has also been used extensively as an agent for protein-protein linkage and hence for fixation. Other uses for glutaraldehyde all of which rely its cross-linking properties include the preparation of tissue xenografts, particularly cardiac valves, chemical sterilisation and disinfection. Glutaraldehyde has an inhibitory effect on catalase5 allowing the selective demonstration of the peroxidase activity of peroxisomes.

MISCELLANEOUS ALDEHYDESAcrolein (acrylic aldehyde) is mainly used in the tanning industry. It produces more cross-links than formaldehyde under optimal conditions but is unpleasant to use and unstable at alkaline pH levels. Acrolein has a tendency to polymerise into disacryl, a solid plastic when exposed to light. It has been employed as a fixative for enzyme cytochemistry as labile enzymes like glucose-6-phosphatase are retained in tissue fixed in 4% acrolein.

Glyoxal (ethanedial, diformyl), malonaldehyde (malonic dialdehyde), diacetyl (2,3-butanedione) and the polyaldehydes are other aldehydes which have been infrequently employed for fixation, mostly for special situations, to retain specific enzymes or proteins for histochemistry. In terms of effectiveness as cross-linking agents glutaraldehyde is the most efficient although acrolein, when present in excess, is nearly as efficient and succinic dialdehyde is also comparable.

OTHER CROSS-RECTING FIXATIVESMany other reagents are available for use as protein cross-linking fixatives although these are not widely employed in histology.

Chloro-s-triazides or cyanuric chloride has been used for the preservation of mucins in rat salivary glands and for immunofluorescence studies.

Carbodiimides are compounds which react with a carboxyl and an amino group with elimination of water to give a peptide and the corresponding urea. This reaction can be used for cross-linking soluble proteins, such as the linkage of small peptides to larger proteins like serum albumin to provide complexes suitable as antigenic stimuli, but they are seldom employed as fixatives.

Diisocyanates are used for introduction of fluorescent labels into proteins.

Diimido esters react rapidly with proteins forming cross-links between amino groups to result in amidines which are stable to acid hydrolysis. These esters have been used as fixatives for electron microscopy9-10 and immunohistochemistry.11

Diethylpyrocarbonate is a compound consisting of diethyl oxydiformate and ethoxyformic anhydrate which is employed for cold sterilisation particularly of alcoholic beverages. This compound reacts with tryptophan to quench background fluorescence induced by aromatic residues. It was first used as a vapour phase fixative for freeze-dried blocks to preserve antigenic determinant sites for proteins and peptides. It has also been proposed as a liquid phase fixative for small blocks, in phosphate buffer at pH 6.0, especially if its solubility is improved with the addition of small quantities of ethanol.

Maleimides, a number of N-substituted bismaleimides, synthesised as sulphydryl reagents, possess mild cross-linking properties for proteins. Lastly, benzoquinone, a compound unsaturated ketone, reacts with amines, amino acids and proteins to give various additional products. It has been used as a fixative for peptide antigens in various endocrine tissues, in both vapour and liquid phase.11

Oxidising agents: metallic ions and complexesMuch less is known of how metallic ions and oxidising agents react with proteins.

OSMIUM TETROXIDE Osmium tetroxide is used for preservation of fine structures in electron microscopy and is effective for small (2-3 mm3) specimens. While vapours 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 ACIDChromic acid (chromium trioxide) is a strong oxidiser 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 tumours. 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.

Protein-denaturing agentsThe structure of proteins is largely dependent on the arrangement of covalent bonds in the

sequence of amino acids forming the peptide chain, and hydrogen bonding between the components of the peptide chain itself and side chains; these forming the primary and secondary structures of a protein respectively. The tertiary structure (the total structure in three dimensions), results from ionic or electrostatic bonds (between the basic and acidic amino-acid residues of peptides), disulphide bonds and hydrophobic bonds (between hydrocarbon-like side chains of leucine, isoleucine, valine, phenylalanine, tryptophan and tyrosine) which are preferentially situated in the relatively water-free interior of the protein molecule. These forces contribute to the exclusion of water from the peptide backbone and are relatively protected from reagents dissolved in the medium. Hydrophobic bonds are weak but as some 30% of the amino acids in a protein will have non-polar side chains, the total effect of hydrophobic bonds is considerable.

METHYL AND ETHYL ALCOHOLAlteration of the structure of proteins brought about by methanol and ethanol is primarily due to disruption of the hydrophobic bonds which contribute to the maintenance of the tertiary structure of proteins. Hydrogen bonds appear to be more stable in methanol and ethanol than in water so that while affecting the tertiary structure of proteins, these alcohols may preserve their secondary structure.

Methanol and ethanol are the only alcohols which have a role as fixatives. Methanol is closely related in structure to water and it competes almost as effectively as the latter for hydrogen bonds. Ethanol is also closely related in structure and both replace water molecules in the tissues, unbound as well as bound, during fixation.

While absolute ethanol preserves glycogen, it can cause distortion of nuclear detail and shrinkage of cytoplasm. If fixation is prolonged, the alcohols remove histones from the nuclei and later extract RNA and DNA.

Methacarn, a 6:3:1 mixture of absolute methanol, chloroform and glacial acetic acid has been used for the preservation of helical proteins in myofibrils and collagen. More recently it has been used as the fixative of choice for the demonstration of intermediate filaments by immunohistochemical techniques.

ACETIC ACIDAcetic 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 ACIDPicric 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 CHLORIDEMercuric 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.

ACETONEAcetone 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.

AdditivesTannic acidTannic acid is a useful addition to the fixation solution as it precipitates of a number of polypeptides and proteins. Tannic acid penetrates tissue easily, imparting high contrast to membranes and staining amorphous material and elastic fibres. It appears to act as a mordant for heavy metal staining and prevents the loss of certain tissue components.

PhenolThe addition of 2% phenol has an accelerating effect on neutral buffered 4% formaldehyde as a fixative with tissue sections showing improved nuclear and cytoplasmic detail, reduced shrinkage and distortion and an absence of formalin pigment. Resin-embedded tissues fixed in the phenol-formaldehyde fixative gave satisfactory preservation of ultrastructural features.

LanthanumLanthanum in its colloidal form has been added to the primary fixative to demonstrate intercellular spaces and cell junctions. It has also been used with Alcian Blue in fixation to demonstrate acid muco-substances on cell surfaces and the addition of lanthanum to glutaraldehyde will prevent the formation of lacunar spaces around chondrocytes in pre-mineralised cartilage, probably by binding to and fixing negatively charged molecules.

LithiumLithium salts have been used to combat volume changes, for example, pre-treatment of glutaraldehyde-fixed tissues with isotonic lithium reduces the shrinkage brought about by ethanol dehydration.

Fixation of specific substances

Glycogen

The use of alcohols has, therefore, been the main method of fixing glycogen in tissues. Earlier fixatives included ice-cold picro-alcohol-formalin or cold alcohol or a mixture of 96% alcohol saturated with picric acid, 40% formalin, and acetic acid. Chemical assays on rat liver have shown 100% ethanol to be clearly superior for the fixation of glycogen. Bouin's fixative is also a useful fixative for glycogen.

LipidsWith 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.

ProteinsThe 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.

MucosubstancesAmong 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 recognised and many fixatives have been suggested to prevent this. Four per cent basic lead acetate was introduced as a fixative for acid heteroglycans and 1% lead nitrate in place of acetate has also been used. Alcoholic 8% lead nitrate, with or without 10% formalin, has been employed for connective tissue glycosaminoglucoronoglycans. Formalin-alcohol mixtures have also been used and calcium acetate has been added to formalin as a cationic precipitate for acidic mucins. 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.

Various cationic dyes have been introduced in attempts to preserve glycosaminoglycans for ultrastructural analysis. Toluidine Blue, Saffranin O, Acridine Orange and more recently a phthalocyanin-like dye, Cuprolinic Blue48 preserve and stain proteoglycans without fixation by glutaraldehyde or formaldehyde. The reaction is based on electrostatic attraction between the positive dye and the polyanionic glycosaminoglycan component.

EnzymesEnzyme activity is best demonstrated histochemically in fresh frozen sections. The most common methods of preserving enzymes for paraffin embedding are fixation in alcohol or acetone usually at 4°C. Alkaline phosphatase activity is retained by both these fixatives when used cold, and the fixing capability of alcohol improves when saturated with sodium -glycerophosphate. Subsequent clearing of alcohol or acetone-fixed blocks in light petroleum (petroleum ether) and embedding in vacuo in low melting point (42-44°C) paraffin improves enzyme preservation further.

The most significant problem in enzyme histochemistry is false localisation due to diffusion of the enzyme. To this end, various fixatives have been described for the optimal preservation of specific enzymes. Formalin-sucrose-ammonia and 1-4% glutaraldehyde have been used for cholinesterases.3,50 The fixation of acid phosphatase can be achieved with formaldehyde and formalin containing 0.1% chloral hydrate will preserve ß-glucuronidase.