Fixation and fixatives
Anthony S-Y Leong

Introduction
Once tissues are removed from the body, they undergo a process of self-destruction or autolysis which is initiated soon after cell death by the action of intracellular enzymes causing the breakdown of protein and eventual liquefaction of the cell. Autolysis is independent of any bacterial action, retarded by cold, greatly accelerated at temperatures of about 30°C and almost inhibited by heating to 50°C.

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 fibre and collagen. By light microscopy, autolysed tissue presents a `washed-out' appearance with swelling of cytoplasm, eventually converting to a granular, homogeneous mass which fails to take up stains. The nuclei of autolytic cells may show some of the changes of necrosis including condensation (pyknosis), fragmentation (karyorrhexis) and lysis (karyolysis) but these are not accompanied by an inflammatory or cellular response. There may be diffusion of intracellular substances of diagnostic significance, such as glycogen which is lost from the cells in the absence of prompt and suitable fixation. Autolysis also causes desquamation of epithelium which separates from its basement membranes.

Bacterial decomposition can also produce changes in tissues that mimic those of autolysis and is brought about by bacterial proliferation in the dead tissue. Such bacteria may normally be present in the body during life such as the non-pathogenetic organisms present in the bowel, or may be present in diseased tissues at the time of death such as in septicaemia.

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 stabilises the cellular and tissue constituents so that they withstand the subsequent stages of tissue processing. Aside from these requirements for the production of tissue sections, increasing interest in cell constituents and the extensive use of immunohistochemistry to augment histological diagnosis has imposed additional requirements. Fixation should also provide for the preservation of tissue substances and proteins. Fixation is, therefore, the first step and the foundation in a sequence of events that culminates in the final examination of a tissue section.

It is relevant to point out that fixation in itself constitutes a major artefact. The living cell is fluid or in a semi-fluid state, whereas fixation produces coagulation of tissue proteins and constituents, a necessary event to prevent their loss or diffusion during tissue processing; the passage through hypertonic and hypotonic solutions during tissue processing would otherwise disrupt the cells. For example, if fresh unfixed tissues were washed for prolonged periods in running water, severe and irreparable damage and cell lysis would result. In contrast, if the tissues were first fixed in formalin, subsequent immersion in water is generally harmless.

A large variety of fixatives is now available but no single substance or known combination of substances has the ability to preserve and allow the demonstration of every tissue component. It is for this reason that some fixatives have only special and limited applications, and in other instances, a mixture of two or more reagents is necessary to employ the special properties of each. The selection of an appropriate fixative is based on considerations such as the structures and entities to be demonstrated and the effects of short-term and long-term storage. Each fixative has advantages and disadvantages, some are restrictive while others are multipurpose. The requirements of a large through-put diagnostic laboratory are also quite different from those of a research laboratory with small numbers of specimens for specialised structural analysis and less requirement for urgency.

Over the years, various classifications of fixatives have been proposed, with major divisions according to function as coagulants and non-coagulants, or according to their chemical nature into three general categories which include alcoholic, aldehydic and heavy metal fixatives. A modification of Hopwood's classification1 shown below will be adopted in this chapter.


Aldehydes, such as formaldehyde, glutaraldehyde.

Oxidising agents: metallic ions and complexes, such as osmium tetroxide, chromic acid.

Protein-denaturing agents, such as acetic acid, methyl alcohol (methanol), ethyl alcohol (ethanol).

Unknown mechanism, such as mercuric chloride, picric acid.

Combined reagents.

Microwaves.

Miscellaneous: excluded volume fixation, vapour fixation.

Fixatives
Aldehydes and other cross-linking fixatives
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 odour, 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 colourless liquid). Formaldehyde is also obtainable in a stable solid form composed of high molecular weight polymers known as paraformaldehyde. Heated paraformaldehyde generates pure gaseous formaldehyde which, when dissolved in water, reverts mostly to the monomeric 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 disputed2 (Fig. 1).

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 ten 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.2 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 recognised 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 oxidises to formic acid, reducing its preserving capabilities such that neutralisation 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 neutralising formalin the resultant solution does not retain its neutral pH and calcium carbonate itself can deposit in tissues, leaving areas of `pseudocalcification'. Phosphate buffers such as sodium phosphate monobasic and sodium phosphate dibasic are effective for the neutralisation of formalin and the pH of the solution produced is stable for many months. Formalin should not be used with chromates because it readily oxidises to formic acid.

A concentrated solution of formalin sometimes becomes turbid on storage through the production of paraformaldehyde which decreases the strength of the solution, but can still be used as a fixative following filtration. Formalin favours 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 end-groups, 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 polymerise. Some favour the polymer as the reactive species while others suggest that pure, monomeric, glutaraldehyde is the best fixative and much less inhibitory to enzymes than is the mixed monomeric-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 monomeric form by removing oligomers, polymers and other impurities through simple shaking with barium carbonate, vacuum distillation or treatment with activated charcoal3 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.4

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 1-4°C. 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 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 ALDEHYDES
Acrolein (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 FIXATIVES
Many 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 glands6 and for immunofluorescence studies.7

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. Diazonium compounds as stabilised salts have been used to reduce diffusion of soluble enzymes from tissue sections and salts such as Fast Garnet GBC, Fast Red B, and Fast Blue R were effective in fixing an otherwise soluble aminopeptidase in rat kidney.8

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 complexes
Much less is known of how metallic ions and oxidising agents react with proteins.

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 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 stabilisation 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 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 ACID
Chromic 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 agents
The 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 ALCOHOL
Alteration 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 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.

Unknown mechanisms
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 litre 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, colourless, 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.

More recently, acetone has been employed as a fixative in the acetone-methylbenzoate-xylene (AMEX) technique.12 This requires overnight fixation of tissues in acetone at -20°C 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.

Microwave (MW) irradiation
MW FOR PRIMARY TISSUE FIXATION
Microwave irradiation for tissue fixation was first introduced by Mayers,13 who reported that direct exposure to MWs generated by a 630-watt heating device, produced satisfactory fixation in both mouse and human post mortem tissues. Subsequently, a more extensive study on the effects of MWs on whole carcases of hairless mice, showed that morphological preservation of various tissues depended upon generation of an optimal temperature for each tissue, which ranged between 70°C and 85°C.14 Heating above or below these temperatures produced various artefacts such as vacuolation and changes in chromatin pattern. Bernard14 was probably the first to indicate that MWs had potential applications in electron microscopy, although he himself was unable to achieve optimal organelle preservation. Heat has been used to induce the partial denaturation of protein but never gained popularity as a method of fixation. This is largely due to the difficulties of controlling conventional forms of heating such as a naked flame, steam, or a water bath, all of which do not result in uniform heating.

MWs are a form of non-ionising radiation, commonly generated by domestic ovens at a frequency of 2.5 GHz. The exposure of dipolar molecules such as water and polar side chains of proteins to the rapidly alternating electromagnetic fields results in oscillation through 180° at the rate of 2.5 billion cycles per second. The molecular kinetics induced result in instantaneous heat which is proportional to the energy flux and continues until radiation ceases. Heating by MWs thus offers a method of overcoming the limitations imposed by the normally poor heat conducting properties of biological tissues. Microwaves of 2.5 GHz penetrate several centimetres into biological material and the heat produced can be controlled by adjustment of the energy levels and the duration of exposure. More recently, it has become recognised that other molecules with an uneven distribution of electrical charge or asymmetrical molecular configuration, such as inorganic material and copper oxides, can also move in the electromagnetic field. As such, MWs have been used to melt inorganic materials and to generate mixed copper oxides for the production of super conductors.

Although heat is currently considered to be the primary factor responsible for many of the effects of MWs in tissue fixation, processing, and staining, the rapid movement of molecules with the electromagnetic flux may itself have a direct role. While heat or thermal energy will increase molecular kinetics and hasten molecular reactions, the rapid movement of molecules directly induced by the oscillating electromagnetic field will give rise to increased collision of molecules and, in turn, accelerate chemical reactions.

In the case of MW accelerated fixation with cross-linking aldehyde solutions, the fixating agents are generally present as oligomers and required degradation to monomers or dimers. The diffusion of these monomers or dimers is enhanced by exposure to MWs. Viscosity, and hence diffusion rates in liquids can vary considerably with temperature, and temperature influences the penetration of tissues by fixing reagents.15 A number of other physical mechanisms have been hypothesised to occur with MW irradiation. Field-induced alterations in macro-molecular hydrogen bonding, proton tunnelling and disruption of bound water may induce alterations in biologic systems. Although the proton energy generated by MWs is too small to alter covalent bonds, low intensity MW fields may readily affect the integrity of non-covalent secondary bonding, including hydrophobic interactions, hydrogen bonds, and van der Waals' interactions that make up the precise steric interactions at the cell membrane.16

Besides the fixation of whole mice14 and rabbit fetuses,17 Leong and Duncis18 investigated the feasibility of using MWs to fix large biopsy specimens and viscera. MWs have limited penetration in solid tissues and temperature gradients of up to 15°C were observed between the surface and core of large pieces of solid tissue such as the spleen, kidney, breast and liver. Because it is impossible to raise the tissue core temperature to an optimal level to accomplish fixation without over-heating the surface, the irradiation has to be done in two phases. Initial irradiation of large specimens such as the stomach, solid organs, and segments of bowel is done primarily to render the tissues sufficiently firm to allow easy handling and dissection. This can be accomplished by completely immersing the specimen in a volume of normal saline and irradiating it to a saline temperature of 68-74°C. This procedure is sufficient to harden the specimen and eliminates the need to pin-out the viscera or to slice the tissue for overnight fixation in formaldehyde as is the convention. Specimens hardened by irradiation have the added advantage of retaining much of their natural colour and pliability. Dissection of lymph nodes in particular, is made easier as they become opaque after irradiation and their pink-tan colour contrasts sharply against bright yellow fat tissue. Because of the limited penetration of MWs and the temperature gradient between surface and deeper regions of large specimens, first stage fixation is uneven and not optimal. Therefore, fixation should be completed after sampling by immersing the 2-3 mm thick tissue blocks in saline and irradiating to a temperature of 50-68°C as described below.18

Domestic MW ovens operating at 2.45 GHz and at 600 Watt output produce satisfactory fixation of most tissues by irradiation in normal saline to a temperature of 50-68°C. When only a small volume of tissue is irradiated, the procedure can be accomplished in about 120 seconds. MWs can thus be used in diagnostic laboratories as the primary method of routine fixation.19-20 Although any domestic oven may be used, those with a rotating plate or carousel give more even distribution of the electromagnetic waves and a digital timer allows greater accuracy. Laboratory-grade ovens are commercially available, but are considerably more expensive and offer no advantage over domestic ovens which are easy to calibrate since the temperature attained is proportional to the duration of electromagnetic flux.20 The availability of temperature probes in some domestic ovens further simplifies operation as these allow a choice of temperature settings.

MW fixation does not have any deleterious effect on special stains. It has also been shown that tissue antigens are often better preserved in MW irradiated tissue than those fixed routinely in 10% formalin and processed in the usual manner.21

The speed with which MWs can accomplish fixation of both large and small biopsy specimens is a major asset. The following procedures can be adopted for large throughput laboratories with requirements of a high speed of turnaround.22
1 Specimens continue to be sent to the laboratory in 10% buffered formalin, a necessary precaution to avoid autolysis which may result from delays and other mishaps that occur during transportation of fresh specimens.
2 Following examination and sampling of these specimens, 2 mm thick blocks are placed in cassettes, completely immersed in normal saline, irradiated to a temperature of 62°C and held at this temperature for 30 seconds (this can be easily set in ovens which have temperature probes). For convenience, 20 cassettes are placed in each of three beakers of saline, equidistantly located at the periphery of the oven's rotating dish. Although morphological preservation is slightly better at higher temperatures, 62°C appears to allow optimal preservation of tissue antigen.
3 Following irradiation, the tissue blocks are processed through cycles of absolute alcohol, chloroform or xylene, and wax in a vacuum-assisted automated processor.


Cycles of 1½ hours and 3½ hours are used, the former for endoscopic and other small biopsies, and the latter for virtually all other tissue blocks. For convenience, an overnight cycle of 16½ hours can still be used if required, details of such cycles being provided in Table 1. During the interim between microwave irradiation and commencement of tissue processing, the tissue blocks can be held in 70% alcohol, Carnoy's solution, 10% buffered formalin, or even normal saline. The first two solutions are particularly useful for tissues that contain large quantities of fat.

The use of such a protocol allows the rapid preparation of good quality diagnostic sections from most specimens. Large pieces of skin and dense tissue, such as myometrium require immersion of approximately 30 minutes in formalin before microwave irradiation, a step which can easily be introduced with little disruption to the procedure set out above.

MICROWAVE FIXATION OF BRAIN
Many of the brain enzymes that catalyse metabolism of compounds such as catecholamines, acetylcholine, GABA, 5-hydroxytryptamine, cyclic AMP, and cyclic GMP are completely and irreversibly decomposed by heat. Irradiation with MWs efficiently arrests such enzymatic activity in brain tissue allowing for more accurate assays of these compounds and, at the same time, hardening the tissues to allow easy dissection.

The irradiation of experimental brain tissue, previously perfused with physiologic saline or formaldehyde has produced excellent tissue sections without the morphological changes that result from autolysis and dehydration and impregnation for routine paraffin processing.23 Compared with cryostat sections of untreated brain, MW-irradiated tissue show fewer changes from freezing-thawing and produce superior sections. Results are better in irradiated tissue following perfusion with saline than those following perfusion with formaldehyde. In addition, the Bodian stain and immunohistochemical staining of neurofilaments in axonal structures are much more distinct in saline-perfused-irradiated tissues.23

MW TO ACCELERATE FIXATION
While MW irradiation may serve as the primary method of fixation, MWs can also accelerate the fixing action (cross-linking) of aldehydes or alcohols. A three-step method using MW irradiation to produce microscopic slices of fresh human brain tissue within one working day has been described.24 The procedure comprises exposure of the whole brain, sprinkled with physiologic saline, to MWs for 30 minutes, followed by irradiation of brain slices for 15 minutes before immersion in 10% formalin for 3½ hours. The final step is a period of MW irradiation in formalin for six minutes.

MW irradiation can similarly be used to accelerate fixation of tissues in glutaraldehyde and other cross-linking agents such as Karnovsky's fixative (0.05% glutaraldehyde and 2% formaldehyde) for electron microscopy (transmission and scanning). For example, tissue samples immersed in 2 µl of the aldehyde fixative can be fixed by irradiating to 50°C (usually requiring 5-10 seconds). This is best achieved by locating the vial of fixative and tissue in the centre 1.5 cm above the carousel on a polystyrene block as the irradiation is most uniform at this site. After removal from the warm solution, the tissue is either stored in 0.1 mol/l sodium cacodylate buffer with 0.02% sodium azide for up to 2 weeks, or immediately processed.25-26 After irradiation in 2.5% glutaraldehyde, the preservation of fine structural features is similar to that of routinely processed tissues.27 Additionally, renal biopsies fixed using MWs and prepared for both light and electron microscopy have been shown to be suitable for immunofluorescence studies, with results equivalent to those of tissues directly snap frozen.28 The use of a MW-irradiation device that maximises the power output and coupling of the electromagnetic energy to the tissue sample, makes possible ultrafast MW fixation with the process accomplished in as brief a period as 26 milliseconds in cross-linking aldehyde solutions.29

Although speed of turnaround is not as important in the preparation of specimens for routine electron microscopy as it is for light microscopy, the ultrastructural examination of fine needle aspiration biopsies does require expedient processing. This aspirated sample is irradiated in 8-10 ml of a 1% glutaraldehyde-4% formaldehyde mixture in cacodylate buffer for 25 seconds in a 650 Watt domestic oven. This is followed by dehydration, embedding in epoxy resin and staining, with a section ready for examination within 2 hours.30 MW stimulated fixation for electron microscopy has the added advantage of preserving of cellular enzymes and proteins which are otherwise difficult to conserve with conventional fixation. These include antigenic sites of rat muscle, chymase and other subcellular structures.31-32

OTHER LABORATORY APPLICATIONS OF MICROWAVE IRRADIATION
An important application of MW irradiation is in the production of vastly improved cell form and structure in cryostat sections. Immersion of freshly cut, frozen sections in Kryofix or Wolman's solution (95% ethanol and 5% glacial acetic acid) followed by exposure to MWs for 15 seconds produces noticeable improvement in the quality of the microscopic image. The cytomorphology is vastly superior to sections conventionally fixed in 95% ethanol, in 10% buffered formalin, or in formalin vapour.

MW irradiation can also be used to accelerate almost every histochemical staining procedure for light microscopy, staining of ultra-thin sections by uranyl acetate and lead citrate for transmission electron microscopy, immunohistochemical procedures, and all the stages of tissue processing.33-34

Excluded volume fixation
The addition of various polymers to a reaction mixture has the effect of 'fixing' small diffusible molecules during the course of the reaction. As the rate at which such molecules diffuse through the polymer is related to its size and concentration, the size of the molecule which can diffuse through the polymer is progressively reduced as the concentration of the polymer rises. Twenty per cent polyvinyl alcohol or 20% polyethylene glycol (Carbowax 6000) have been used but mostly in an experimental situation.

Vapour fixation
Vapour as opposed to liquid fixation was originally used to retain soluble materials in situ by converting them to insoluble products before contacting with water or non-aqueous solvents. Various chemicals which act as vapour fixatives include aldehydes (formaldehyde, glutaraldehyde and acrolein), osmium tetroxide, chromyl chloride, ethanol, diethylpyrocarbonate, benzoquinone, and diacetyl; the most common being formaldehyde, osmium tetroxide, and perhaps alcohol. The most important application of vapour fixation has been the use of formaldehyde at elevated temperatures for the conversion of catecholamines and 5-hydroxy-tryptamine in freeze-dried tissue to produce fluorescent condensation products. This method35 highlights the ability of highly reactive fixative vapour to capture and render insoluble otherwise highly soluble, low molecular weight compounds. The usual source of monomeric formaldehyde is heated paraformaldehyde and the technique can also be applied to frozen sections.

Osmium tetroxide at 37°C produces a vapour pressure which is sufficient to allow very rapid penetration into freeze-dried blocks of tissue and exposure for 1 hour or less is usually adequate.

Ethanol vapour at 60°C has a pronounced denaturing effect on freeze-dried tissues and polysaccharides like glycogen become less soluble. There is, however, no real application for this technique in diagnostic pathology.

Freeze-drying
Stein et al36 described a method of freeze drying at -45°C, 10-2 torr in the presence of phosphorus pentoxide for up to 48 hours before embedding in paraffin, for the preservation of labile lymphocyte antigens. While effective, the method is cumbersome and requires special equipment. The resultant tissues have also proved to be difficult to section and often display suboptimal morphology. Another method, freeze substitution using low temperature plastic embedding, avoids the need for fixation and retains tissue morphology and immunoreactivity.37

Postfixation
In situations where, for various reasons, the primary fixation process may not be optimal, the wet tissue may be subjected to post fixation or secondary fixation. For example, tissues fixed in buffered formalin may be subjected to further fixation with a mercuric chloride-formaldehyde solution for a period of several hours to improve staining of sections. Other post fixatives such as a mercuric chloride-picric acid-formaldehyde mixture may be used. The main disadvantages of post fixation is the extra time and costs involved as well as the toxicity of the mercuric solution.

Post fixation in osmium tetroxide is common in electron microscopy for tissue blocks previously fixed in glutaraldehyde. Osmium tetroxide improves the staining of cell membranes although the actual mechanism involved is not completely known.

Fixation mixtures
A mixture of osmium tetroxide and glutaraldehyde has been shown to be useful for neutral fat and fine structural localisation of acid phosphatase. Other mixtures such as osmium tetroxide-zinc iodide post fixation have been used for delineating synaptic vesicles and Langerhans' cells, and glutaraldehyde and carbodiimide have been used as a primary fixative for electron microscopic immunohistochemistry. McDowell and Trump38 developed a general purpose fixative for both light and electron microscopy comprising a mixture of formaldehyde and glutaraldehyde.

Additives
Tannic acid
Tannic 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. Its action is independent of aldehyde groups in the tissues and its mordanting function is dependent on carboxyl and at least one hydroxyl on the benzene ring. Tannic acid has been added to formaldehyde-glutaraldehyde solutions, allowing endogenous lactoperoxidase in mammary tissue to be demonstrated, and it may also increase the retention of lipids in tissues. Tannic acid diffuses easily through tissues, penetrating and enhancing the density of damaged cells allowing their differentiation from intact cells both by light and electron microscopy.

Phenol
The 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.39 Resin-embedded tissues fixed in the phenol-formaldehyde fixative gave satisfactory preservation of ultrastructural features.

Heavy metal solutions
Transitional metal salts such as zinc can be potent protein precipitants, forming insoluble complexes with polypeptides. Zinc formalin has been proposed therefore as a fixative to enhance immunostaining40 and antigen preservation.41 There is some suggestion that post fixation in zinc sulphate may also improve immunostaining.42 The introduction of zinc formalin (1% zinc sulphate in 3.7% unbuffered formalin) into automated tissue processors has produced no deleterious effects or damage.41

Lanthanum
Lanthanum 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.

Lithium
Lithium 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.

Detergents
Detergents have been used with the primary fixative to enhance the effect of some other reagent used simultaneously or subsequently in the overall technique. For example saponin and Novidet aid the subsequent use of antibodies whilst Triton X-100 increases tissue permeability for Ruthenium Red. It should be noted that detergent generally has a deleterious effect on cytological detail and its application is limited to only specific situations.

Factors involved in fixation
Temperature
While the fixation of specimens for standard histology is generally carried out at room temperature for convenience, for electron microscopy and some histochemical procedures, the temperature for fixation is usually 0-4°C. At this lower temperature range autolysis is slowed down, as is the diffusion of various cellular components, allowing a more life-like appearance of the tissues. However chemical reactions, including those involved in the fixation process are frequently more rapid at higher temperatures. The use of heat for fixation in microbiology and for blood films is well known and the heating of formalin to temperatures of 60-70°C can be used for the rapid fixation of very urgent biopsy specimens, although the risk of tissue distortion is increased.

Size of specimens and penetration of fixative
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. The penetration of fixatives into tissues can be determined from the following equation based on the laws of diffusion:

d = KÖ t

Where
d is the depth penetrated
t represents time
K (a constant) is the coefficient of diffusibility of the fixative and is specific for each fixative. It represents the distance in millimetres the fixative has diffused into the tissue at 1 hour.

An indication of the relative diffusibility of various fixatives in a uniform tissue such as the liver, at room temperature are given in Table 2. More recent work suggests that in human tissues, the rate of fixative diffusion may even be lower.43

Changes in volume
The mechanisms involved in volume changes in tissues with fixation are not well understood. Those suggested include inhibition of respiration, changes in membrane permeability and changes in ion transport through membranes. Some intercellular substances such as collagen may swell when they are fixed. There are also changes in volume which occur subsequently during dehydration and paraffin embedding. It has been estimated that tissue fixed in formaldehyde and embedded in paraffin wax shrinks by 33% and this is evident when paraffin embedded sections are compared with frozen sections, the latter showing larger nuclei and cells.

pH and buffers
The hydrogen ion concentration varies between fixatives, but in general, the pH should be kept in the physiological range, between pH 6-8. This can be maintained by buffer systems, the most common being phosphate, s-collidine, bicarbonate, Tris, veronal acetate and cacodylate. The chosen buffer should not react with the fixative as this will reduce both the buffering power and the function of the fixative. Furthermore, the buffer should not inhibit enzymes or react with the incubation medium if histochemical procedures are to be performed.

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.

Concentration of fixatives
Some fixatives are effective within a range of different concentrations, for example, glutaraldehyde which may be used as a 4% solution is effective as low as 0.25%, provided the pH is maintained in the physiological range. This allows large volumes of aldehyde fixatives to be prepared at one time. (The presence of a buffer may cause polymerisation of the aldehyde with a consequent decrease in its concentration). There is, however, some variation in the staining intensity of tissues with variation in the concentration of fixative.

When vehicles other than buffer and water are added the effectiveness of the fixative solution may be altered. For example, some salts have denaturing effects while others such as ammonium sulphate and potassium dihydrogen phosphate strongly stabilise proteins. Sodium chloride and sodium sulphate are used in various fixative mixtures containing mercuric chloride because sodium chloride may increase the binding of mercuric chloride to the amino groups of proteins and may also dissolve coagulated proteins.

Duration of fixation
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 oxidising fixatives can degrade tissues by oxidative cleavage of proteins and loss of peptides.

Fixation of specific substances
Glycogen
A variety of glycogens occur naturally and show different degrees of polymerisation. The less highly polymerised 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 polymerised glycogens are retained with a wide variety of fixatives as well as alcohol-containing 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.

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.

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 cholesterol44-45 and Malachite Green included with glutaraldehyde or Karnovsky's fixative retains various lipids such as phospholipids, fatty acids, glycolipids and choline plasmalogen.46 Imidazole introduced into the post osmication of glutaraldehyde-fixed tissue demonstrates unsaturated fatty acids and phospholipids as electron-dense deposits.47

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 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.

The reactions of cetylpyridinium salts (with acid glycosaminoglycans) and acridines to form insoluble complexes are superior to formalin, Carnoy's solution and formalin-alcohol mixtures, for the preservation of heteroglycans. The most successful method for preserving all types of mucin is freeze drying followed by hot formaldehyde vapour.

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.

Nucleic acids and nucleoproteins
The nucleic acids exist in many different states of polymerisation 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.

Biogenic amines
The biogenic amines include two main groups, the catecholamines, adrenalin and noradrenaline, and the indolalkylamines, dopamine, DOPA and 5-hydroxytryptamine. The chromaffin reaction may be demonstrated by fixing the tissue in a solution of formalin with sodium acetate and potassium dichromate. The biogenic amines can also be demonstrated by formaldehyde-induced fluorescence.

Glutaraldehyde at pH 7.2 - 7.4 precipitates noradrenaline but adrenalin dissolves into the solution unless 1.5% of potassium dichromate is added. A more effective method of retaining biogenic amines for ultrastructural examination is to use a three-stage fixation procedure.49 This comprises primary fixation in a mixture of 1% glutaraldehyde, 0.4% formaldehyde, sodium chromate and potassium dichromate followed by storage for 18 hours in a mixture of sodium chromate and potassium dichromate, and finally, post fixation in 2% osmium tetroxide, sodium chlorate and potassium dichromate.

Enzymes
Enzyme 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 a -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.

Agonal changes and fixation artefacts
Ideally, tissues should be fixed immediately and completely from the living state, but this cannot be achieved for human tissues. As most tissues are removed surgically, the organ or tissue is relatively anoxic for some period because of anaesthesia and the placement of surgical clamps and ligatures to stop bleeding. Furthermore, when the tissue is placed in fixative, there is a latent period before adequate penetration of the tissue. Anoxic changes including damage to mitochondria are noticeable ultrastructurally within 10 minutes and enzymes such as those concerned with oxidative phosphorylation are lost within an hour. There is also variation within the tissue as cells in the centre of the block suffer more anoxia due to delays in penetration of the fixing solution. Anoxic changes occur more rapidly at room temperature than in the cold and thus are exaggerated with post mortem material as the body remains at room temperature for variable periods before it is transferred to the mortuary and refrigerated. Furthermore, as the autopsy may not be conducted for several hours or days after death, some degree of autolysis invariably occurs.

While one of the major aims of fixation is preservation of tissues in as life-like state as possible, it is important to appreciate that fixation itself may cause certain artefacts. Expansion and shrinkage of tissues during fixation have been previously discussed. Another important artefact relates to the movement of unfixed material so that organelles and other subcellular structures may be falsely localised at sites where they do not belong. For example, the vesicles which are commonly seen fused with the fibroblast cell membrane are not seen with freeze fracture techniques, and it is felt that the vesicles exist in the subjacent cytoplasm and are induced to fuse with the cell membrane by glutaraldehyde fixation. Glutaraldehyde preserves the interdigitations of cell membranes whereas osmium tetroxide causes their breakdown into vesicles. It has been noted that tight junctions in rat liver and small intestine vary according to the method of preparation, and tight junction fibrils are produced by the cross-linking of junctional proteins by glutaraldehyde. Unfixed haemoglobulin diffuses from red blood cells to the periphery of a block because of the relatively slow penetration of glutaraldehyde. This illustrates the ability of large molecules to diffuse during fixation and also underlines the necessity to use only small blocks of tissue.

Not only may large molecules move within a block but materials may diffuse from the tissue altogether. During fixation this occurs with both large and small molecules including inorganic ions and cofactors for enzymes. Biogenic amines, for example, are stored in membrane-bound granules in the cells of the adrenal medulla, neurons and other neuroendocrine organs. Fixation denatures chromogranin, an associated protein, with the release of the biogenic amines and ATP. Unless the amines can be fixed in some way such as by precipitation, they may be lost from the tissue. Indeed, this diffusion can be seen macroscopically when adrenal tissue is placed in iodate. As catecholamines diffuse from the tissue into the fixing solution, they react with iodate and can be seen as a haze of red aminochromes. The opposite situation may also occur with false fixation of extraneous material within the tissue. This is particularly so when employing radioactive labelled amino acids, sugars, thymidine and uridine.

Chemical changes caused by fixation may give false histochemical reactions in the tissue. For example, glutaraldehyde will add carbonyl groups to tissues and these will react with Schiff's reagent. In removing excess mercuric chloride after fixation, substances such as histidine, tyrosine and mercaptides may also be removed.

Lastly, if the fixative employed was chosen for the preservation of a specific substance, it is likely that many other cellular substances will be lost as there is currently no single fixative that will preserve all tissue substances. The loss of components may be due to their lack of reaction with a fixative and subsequent removal during processing or may result from degradation by the fixative as in the case of small molecules such as biogenic amines; while glutaraldehyde will precipitate noradrenaline, adrenalin is lost from the tissues. Formaldehyde at pH 7 causes the loss of about 60% of catecholamines from chromaffin granules and 5-hydroxytryptamine is lost during fixation in formaldehyde and glutaraldehyde51. Formalin is an inadequate fixative for the proteoglycans of many pituitary glands and many neuropeptides such as luteinizing hormone-releasing hormones are ethanol soluble and may be lost during dehydration. The difficulty in retaining lipids in sections is well recognised and the loss of mucosubstances during fixation is also well documented. Enzymes may be released during fixation and ions may be lost from tissues. Prolonged fixation in formaldehyde results in the loss of water soluble materials particularly when fixation exceeds 6 hours. Proteins are degraded by osmium tetroxide.

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 emphasise 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% glutaraldehyde 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. Microwave irradiation is a physical modality which provides an alternative method of primary fixation. It is rapid, cheap, safe and clean and can also be employed as a means of stimulating accelerated fixation by other known fixatives and mixtures.

Formulations for various fixtives
The details provided relate to commonly used fixatives. Many variations are available and more specialised fixative solutions are not provided.

Formaldehyde solutions
10% neutral buffer formalin (4% formaldehyde)
REAGENTS REQUIRED
1 40% formaldehyde 100 ml
2 Distilled water 900 ml
3 Sodium dihydrogen orthophosphate 4 g
4 Disodium hydrogen orthophosphate (anhydrous) 6.5 g


METHOD
Prepare, using quantities indicated. Fixation time: 24-72 hours.

Baker's formol-calcium (modified)
REAGENTS REQUIRED
1 40% formaldehyde 100 ml
2 Distilled water 900 ml
3 10% calcium chloride 100 ml
4 7 g of cadmium chloride is sometimes added to the mixture


METHOD
Prepare, using quantities indicated. Fixation time: 16-24 hours.

Formol saline
REAGENTS REQUIRED
1 40% formaldehyde 100 ml
2 Sodium chloride 9 g
3 Tap water 900 ml


METHOD
Prepare, using quantities indicated.

Alcoholic formaldehyde
REAGENTS REQUIRED
1 40% formaldehyde 100 ml
2 95% alcohol 900 ml
3 0.5 g calcium acetate may be added to this mixture to ensure neutrality


METHOD
Prepare, using quantities indicated. Fixation time: 16-24 hours.

Paraformaldehyde
REAGENTS REQUIRED
1 Solution A
2.26% sodium dihydrogen orthophosphate 41.5 ml
2.52% sodium hydroxide 8.5 ml
Heat to 60°C-80°C in a covered container


2 Paraformaldehyde 2 g


METHOD
1 Add paraformaldehyde to solution A, stirring until the mixture is clear.
2 Filter and cool. Adjust pH to 7.2 - 7.4.
3 Prepare fresh for use (duration of fixation depends on size of specimen and whether for light or electron microscopy).



Buffered formaldehyde-glutaraldehyde 200 mOsm38
REAGENTS REQUIRED
1 Sodium dihydrogen orthophosphate 1.6 g
2 Sodium hydroxide 0.27 g
3 Distilled water 88 ml
4 40% formaldehyde 10 ml
5 50% glutaraldehyde 2 ml


METHOD
Prepare, using quantities indicated. Fixation time: 16-24 hours.

Alcoholic fixatives
Carnoy's fixative
REAGENTS REQUIRED
1 Absolute ethanol 60 ml
2 Chloroform 30 ml
3 Glacial acetic acid 10 ml


METHOD
Prepare, using quantities indicated. Fixation time: 1-5 minutes.

Methacarn
REAGENTS REQUIRED
1 Absolute methanol 60 ml
2 Chloroform 30 ml
3 Glacial acetic acid 10 ml


METHOD
Prepare, using quantities indicated. Fixation time: 5-6 hours.

Wolman's solution
REAGENTS REQUIRED
1 Absolute ethanol 95 ml
2 Glacial acetic acid 5 ml


METHOD
Immerse frozen section in solution and microwave at 650 watts for 15 seconds.

Acetic alcohol formalin
REAGENTS REQUIRED
1 40% formaldehyde 10 ml
2 Acetic acid 5 ml
3 Ethanol 85 ml


METHOD
Prepare, using quantities indicated. Fixation time: 24 hours at 4°C.

Picric acid fixatives
Rossman's fluid
REAGENTS REQUIRED
1 100% ethanol saturated with picric acid 90 ml
2 Neutralised commercial formalin 10 ml


METHOD
Prepare, using quantities indicated. Fix for 12-24 hours and wash very well in 95% ethanol.

Gendre's fluid
REAGENTS REQUIRED
1 90% ethanol saturated with picric acid 80 ml
2 40% formaldehyde 15 ml
3 Glacial acetic acid 5 ml


METHOD
Prepare, using quantities indicated. Fixation is normally for 4 hours, followed by washing in 80%, 95% and 100% ethanol.

Bouin's fluid
REAGENTS REQUIRED
1 Saturated aqueous picric acid solution 75 ml
2 40% formaldehyde 25 ml
3 Glacial acetic acid 5 ml


METHOD
1 Prepare, using quantities indicated. Fixation may vary from a few hours to 18 hours.
2 Washing with 70% ethanol after fixation will remove most of the yellow colour. Sections can also be washed after removal of paraffin wax.


Mercuric fixatives
Buffered formaldehyde sublimate
REAGENTS REQUIRED
1 Mercuric chloride 6 g
2 Distilled water 90 ml
3 Sodium acetate 1.25 g
4 40% formaldehyde 10 ml


METHOD
Prepare, using quantities indicated. Fixation time: 16-18 hours.

Zenker's fluid
REAGENTS REQUIRED
1 Distilled water 950 ml
2 Potassium dichromate 25 g
3 Mercuric chloride 50 g
4 Glacial acetic acid 50 g


METHOD
Prepare, using quantities indicated. Fixation is normally for 4-24 hours followed by an overnight wash.

Helly's fluid
REAGENTS REQUIRED
1 Solution A
Distilled water 1000 ml
Potassium dichromate 25 g
Sodium sulphate 10 g
Mercuric chloride 50 g


2 Solution B
40% formaldehyde 50 ml


METHOD
Add solution A to solution B immediately before use.

B5 fixative
REAGENTS REQUIRED
1 Stock reagent A
Mercuric chloride 60 g
Sodium acetate 12.5 g
Distilled water l

2 Stock reagent B
10% buffered neutral formalin

METHOD
To prepare a working solution mix 90 ml stock reagent A with 10 ml stock reagent B. Fixation time: 5-8 hours.

Susa fluid
REAGENTS REQUIRED
1 Distilled water 80 ml
2 40% formaldehyde 20 ml
3 Glacial acetic acid 4 ml
4 Trichloroacetic acid 2 g
5 Mercuric chloride 4.5 g
6 Sodium chloride 0.5 g


METHOD
Prepare, using quantities indicated. Fixation time: 12 hours.

TECHNICAL NOTES
Mercuric chloride is usually combined with other fixatives as it penetrates poorly and produces tissue shrinkage (formaldehyde is the additional fixative in the above mixture). Fixatives containing mercuric chloride produce a black precipitate of mercury which can be removed by placing sections in 0.5% iodine solution in 70% ethanol for 5-10 minutes, followed by rinsing in water, decolourisation in 5% sodium thiosulphate for 5 minutes and rinsing in water again.

Fixation of specific substances
Glycogen
Alcoholic fixatives such as Rossman's fluid, Gendre fluid and ethanol fix glycogen.

LIPIDS
Elftman's fixative
REAGENTS REQUIRED
1 Mercuric chloride 5 g
2 Potassium dichromate 2.5 g
3 Water 100 ml


METHOD
Prepare using quantities indicated. Fixation time: 3 days at room temperature.

Swank and Davenport's fixative
REAGENTS REQUIRED
1 1% potassium chlorate 60 ml
2 40% formaldehyde 12 ml
3 Acetic acid 1 ml


METHOD
Prepare, using quantities indicated. Fixation time: 6-10 days.

Other fixatives for lipids include Baker's formol-calcium for phospholipids, and osmium tetroxide for unsaturated lipids.

MUCOSUBSTANCES
Lillie's alcoholic lead nitrate
REAGENTS REQUIRED
1 Lead nitrate 8 g
2 40% formaldehyde 10 ml
3 Water 10 ml
4 Ethanol 80 ml


METHOD
Prepare, using quantities indicated. Fixation time: 24 hours at room temperature.

Cetylpyridinium chloride (C.P.C.)
REAGENTS REQUIRED
1 40% formaldehyde 10 ml
2 Cetylpyridinium chloride 0.5 g
3 Water 90 ml


METHOD
Prepare, using quantities indicated. Fixation time: 48 hours.

Lead subacetate-ethanol fixative
REAGENTS REQUIRED
1 Lead subacetate 1 g
2 Ethanol 50 ml
3 Water 50 ml
4 Acetic acid 0.5 ml


METHOD
Prepare, using quantities indicated. Fixation time: 24 hours.

BIOGENIC AMINES
Potassium dichromate-acetate-formaldehyde fixative
REAGENTS REQUIRED
1 5% potassium dichromate solution 60 ml
2 1 mol/l sodium acetate 10 ml
3 40% formaldehyde 12 ml
4 Distilled water 18 ml


METHOD
Prepare, using quantities indicated. Fixation time: 18 hours.