Tissue processing

Leigh Winsor INTRODUCTION

Stabilised tissues must be adequately supported before they can be sectioned for microscopical examination. Whilst they may be sectioned following a range of preparatory freezing methods, tissues are more commonly taken through a series of reagents and finally infiltrated and embedded in a stable medium which when hard, provides the necessary support for microtomy. This treatment is termed tissue processing. ethods have evolved for a range of embedding media and applications !Table."#. $re% eminent amongst these is the paraffin wax method, discussed here in detail, which is considered to be the most suitable for routine preparation, sectioning, staining and subsequent storage of large numbers of tissue samples. The quality of structural preservation seen in the final stained and mounted section is largely determined by the choice of fixative and embedding medium. &uring tissue processing loss of cellular constituents and shrinkage or distortion should be minimal. 'fter fixation, post%fixation and preparatory procedures, the four main stages in the paraffin method are dehydration, clearing, infiltration and embedding.

Tissue sampling and identification

Tissue sampling generally follows standard protocols",( established by each laboratory for particular species and categories of specimens. Tissue blocks for processing should be as thin as is consistent with the purpose for which they are required, usually "%( mm thick for urgent specimens and rapid processing) *%+ mm for routine material processed overnight. Specimens should not be tightly packed into processing cassettes or containers, but should have sufficient free space to facilitate fluid exchange. Small specimens and tissue fragments are processed in fine mesh containers, wrapped in lens tissue, sandwiched between sponge biopsy pads or more safely, double embedded in agar%paraffin wax. Specimens are generally identified by a numbering system that is not bleached by subsequent fluid and solvent treatment. ,xamples include !a# a numbered card label generated by computer%printer, or handwritten in soft lead pencil or waterproof ink !b# colour coded plastic cassettes, machine or manually labelled. Tissue marking and orientation arking facilitates identification and correct orientation of particular tissue pieces or surfaces during embedding and subsequent microscopical examination. Tissue blocks are simply marked by cutting a notch on the reverse side of the block face to be sectioned, or by trimming the block to a particular shape. -owever dye marking is preferred for certain surgical specimens, small tissue pieces, and for serial sectioning orientation. TISSUE MARKIN SU!STANCES .riteria* for the selection of a suitable tissue marker are/

the marking substance must be relatively insoluble in fixative, processing reagents and embedding medium. it must survive fixation and processing and not result in unacceptable contamination of the reagents and other tissues processed simultaneously. it must remain on the surface of the specimen and not penetrate tissue. it should not react unfavourably with histological stains and must be clearly identifiable both macroscopically and microscopically. for some purposes it may be important that the marker is either radiolucent or radio%opaque.

Tissue markers are applied to the surface of the specimen using disposable swabs and allowed to dry. India ink provides good black macro and microscopic marking, is resistant to processing, but takes "+%*0 minutes to dry, and may spread beyond the marked area. Silver and gold inks are not recommended as they are solvent soluble1. Sil"er nitrate !stick# provides a brown%black mark resistant to processing. 'queous or alcoholic silver nitrate solutions behave like 2ndia ink and are not recommended. Artists# grade pigments are radio%opaque, processing resistant and provide good macro and microscopic contrast. $repare by finely grinding pigment !+03 w4v# to a thin paste in acetone1,and store in tightly stoppered containers. These markers dry in "+%*0 minutes. $articulate pigments, 53 pigment w4v in (13 gelatine solution*, dry in less than + minutes, or in about "0 seconds on chilled specimens. $aprika, turmeric, henna, 2ndia ink, and 6ismark brown are all inexpensive, strongly coloured processing%resistant pigments with distinctive microscopic particle morphology. Alcian %lue, "3 aqueous solution, is a rapid and reliable stain for marking resection margins of fixed breast+ and other biopsies. The specimen is dipped into the stain for a few seconds then blotted dry. Sufficient dye remains to mark resection margins. Eosin& Er't(rosin and Rose !engal, "%(3 aqueous, are used to stain small translucent specimens. Tissues are stained for + minutes, rinsed in water then processed. 'lthough some dye is lost in the dehydration alcohols, sufficient remains to render the tissues visible. 'lternatively dye is incorporated in the 7+3 ethanol dehydrant, and tissues stained during the routine dehydration step. Tissue marking dyes are available commercially and have been favourably evaluated8.

Completion of fi)ation
Tissues should be fixed before processing is initiated. $oorly fixed tissues are inadequately protected against the physical and chemical rigours of processing. Strategies commonly employed to ensure complete fixation of tissues include/

microwave irradiation of biopsy specimens in normal saline.9%7 continuing fixation on the tissue processor with one or more changes of the routine fixative, often at elevated temperatures of 10:.%80:.. secondary fixation of tissues in formol sublimate on the tissue processor"0, or in an alcoholic fixative which will complete fixation whilst initiating dehydration. fixing in buffered phenol%formaldehyde p- 9.0 and p- +.+ sequence at 10:."".

$ost fi)ation procedures

;n completion of fixation, tissues fixed in certain reagents must undergo special treatment. *i)ati"es containing dic(romate and c(romium trio)ide &ichromates and chromium trioxide are reduced to insoluble green%brown chromic oxide in the higher alcohols and in dioxane. Tissues must be washed for 5%"( hours in running water before transferring to 803%903 ethanol or dioxane. *i)ati"es containing p(osp(ate $hosphate salts precipitate in alcoholic solutions stronger than 903 ethanol, in dimethoxy propane, and in diethoxy propane. 2f they are deposited within tissues the precipitate can cause sectioning difficulties"(. Tissues are rinsed free of fixative with water and processing initiated in 803%903 ethanol. *i)ati"es containing picric acid Tissues fixed in non%alcoholic picric acid%based fixatives are washed in repeated "%* hourly changes of +03%903 ethanol until the supernatant is faintly yellowish or clear. This may take (%* days. Specimens fixed in alcoholic picric acid fluids are washed in 503%703 ethanol, as anhydrous conditions must be maintained. $icric acid retained in tissues can impede wax infiltration and exacerbate static electrification of ribbons during sectioning. 2t also has an adverse affect on stored wax embedded tissues"*. *i)ati"es containing urea Tissues fixed in urea containing fluids are washed overnight before transfer to 13 formaldehyde solution for storage. <rea complexes with formaldehyde to form insoluble urea%polymer pigments"*. Specific fi)ati"e re+uirements .arnoy fixed tissues are near%anhydrous and are placed directly in absolute ethanol or in alcohol%transition solvent. -eidenhain=s S<S' fixed tissues are transferred directly to 7+3 ethanol, as trichloroacetic acid fixed collagen swells in aqueous solutions. Tissues fixed in osmium tetroxide%based fixatives are washed for + hours in running water and dehydration initiated in *03 ethanol. ;smium tetroxide is reduced to black osmium in ethanol.

$rinciples of tissue processing

Tissue processing is concerned with the diffusion of various substances into and out of stabilised porous tissues. The diffusion process results from the thermodynamic tendency of processing reagents to equalise concentrations inside and outside blocks of tissue, thus generally conforming to >ick=s ?aw/ the rate of solution diffusion through tissues is proportional to the concentration gradient !the difference between the concentrations of the fluids inside and outside the tissue# as a multiple of temperature dependant constants for specific substances. >rom this it can be seen that the significant variables in tissue processing are the operating conditions, particularly temperature, the characteristics and concentrations of the reagents and the properties of the tissue. T(e tissue Tissue porosity has a ma@or impact on processing, subsequent microtomy and staining. $orosity at an ultrastructural level is determined by the nature and composition of the tissues, and the effects of fixatives, modifiers, and processing reagents to which the tissues are sub@ected. Tissue porosity involves natural and artefactual pores, and the swelling and shrinkage of the biopolymer matrix !>ig. "#"1%"+. ,ven after fixation cell surfaces continue to act as osmotic membranes"+. 2rrespective of the fixative used, all tissues undergo limited shrinkage and hardening during dehydration, clearing and infiltration"+%"5 as well as staining and mounting"+. -ardening generally results from tissue shrinkage, accompanied in most cases by decreased tissue porosity"7. >atty tissues usually require extended processing as lipids, such as myelin in brain tissues and general body fats, inhibit the diffusion of processing reagents. *I,ATION 2n general there is a tendency for tissues fixed in reagents that cause little initial shrinkage to undergo a greater degree of shrinkage during dehydration. .onversely tissues fixed in substances which cause considerable initial shrinkage, contract less during dehydration"9,(0,(". Aon%protein coagulant fixatives such as formaldehyde, result in little ultrastructural tissue damage"1. -owever these agents tend to swell tissues"+, and generally fail to give adequate protection from shrinkage and hardening during subsequent processing to paraffin wax !>ig. (#"+,"9,(0%(*. Aon%fixative salts, such as calcium chloride, incorporated in formaldehyde fixatives further stabilise tissues and as a consequence reduce processing% induced tissue shrinkage"+,"8. $rotein%coagulant fixatives such as ethanol, mercuric chloride or picric acid, shatter tissue ultrastructure"1 but this may increase porosity. Tissues may shrink during fixation but are protected against further significant contraction and hardening during processing !>ig. (#"+,"8,"9,(0%(*. Secondary fixation of formaldehyde fixed tissues with coagulant fixative mixtures including formal sublimate, -elly=s or 6ouin=s fluids, enhances the response of these tissues to processing, sectioning and staining"0.

&uration of fixation determines the extent of tissue stabilisation and consequently porosity and influences reactivity in histological and immunohistochemical procedures. <nderfixed tissues are inadequately protected against processing reagents and exhibit a range of artefacts"*, including those associated with secondary fixation by the dehydrant. $rolonged exposure to primary and secondary fixatives during processing may impair tissue reactivity, particularly in immunohistochemical investigations. SO-.ENT E**ECTS ?oss of certain substances from tissues during processing may also indirectly affect tissue porosity and result in shrinkage(1. ,ven where the ultrastructure has not undergone disruption, tissue porosity can be increased for example, by the dissolution of lipid%rich structures such as membranes and fat droplets by processing solvents, which can then result in shrinkage(+. Some processing fluids such as glycerol for example, also affect tissues by increasing softness but decreasing porosity"7 resulting in protracted clearing and infiltration times thus negating the original softening action. ;ther reagents including cedarwood oil"7 maintain both softness and porosity and facilitate subsequent processing steps. TISSUE MODI*IERS Tissue modifiers such as phenol swell unfixed collagen and elastic fibres"1, enhance protein polymer formation in formaldehyde fixed tissues"",(8 coagulate proteins, and probably maintain or promote tissue porosity. These phenomena underlie the use of phenol and other surfactants to stabilise and soften hard tissues during fixation"",(8%*0 and processing"",(8,(7,*"%*(. DENSIT/ AND T0ICKNESS Bariable tissue density affects infiltration and subsequent microtomy**. Spongy, parenchymatous tissues are usually more rapidly infiltrated than hard and dense tissues. 6lock thickness also influences the rate of reagent diffusion and hence processing time. Tissue thickness should be optimised for particular processing schedules, or alternatively processing times are ad@usted to accommodate thick, thin or large tissue blocks. $rocessing reagents The chemical and physical properties of reagents which influence processing include polarity, concentration, miscibility with water, solvents and embedding media, evaporation rate, and viscosity !Tables (, *#. Thermal conductivity, heat capacity, boiling point and the electromagnetic conductivity of reagents are particularly important in microwave%stimulated processing*1. $O-ARIT/ To minimise tissue distortion there should be a gradual change in polarity of the processing fluid from highly polar aqueous fixatives and solutions to the embedding medium which is usually non%polar !hydrophobic#*+. Tissues generally shrink when transferred to a fluid of relatively lower polarity*+.

CONCENTRATION 2f the concentration gradient between fluid inside and outside the tissue is too high, rapid reagent diffusion and accompanying strong diffusion currents have the potential to shrink and disrupt tissues. >or this reason specimens are almost always processed through a graded series of reagents of increasing concentration, the more delicate the tissues, the closer the gradations. MISCI!I-IT/ $rocessing reagents which are miscible with water and with the embedding medium reduce the number of processing stages and are termed universal solvents. To avoid severe tissue shrinkage from concentration and polarity effects, they are often employed in a graded series. any transition solvents, for example xylene, are extremely water%intolerant, and are immiscible with hydrated alcohols. .ouplers such as phenol mixed with a transition solvent permit clearing from 903%7+3 alcohols*8. ;riginally couplers were employed to overcome difficulties with hydrated higher alcohols. -owever they are now used in processing yolky or blood%filled tissues which harden excessively if fully dehydrated in absolute ethanol, and complement the use of tissue modifiers. E.A$ORATION RATE The evaporation rate, rather than the vapour pressure or boiling point of a solvent, is the best predictor of the rate of elimination of a substance from molten infiltrating wax. Solvents with high evaporation rates are the most readily vaporised and are less likely to contaminate the infiltration medium. .ISCOSIT/ Biscosity is the internal friction of a particular substance which affects rate of flow through tissues and is inversely proportional to temperature. 2t is particularly important in the clearing and infiltration stages of processing. Substances with high molecular weight, such as some transition solvents and waxes, have high viscosities and diffuse through tissues more slowly than, for example, the lower molecular weight, lower viscosity dehydrant alcohols. 2f tissue shrinkage or swelling is to be avoided when the specimen moves from one processing step to the next, the fluid already in the tissues must diffuse outward through the tissue pores at the same rate as the fresh medium diffuses inwards. 2f the viscosity differential between fluid inside and outside the tissue is too great, shrinkage will result. -ence slow and gradual processing of tissues is necessary when viscous reagents are used !for example in nitrocellulose embedding methods#. EM!EDDIN MEDIA 2nfiltrating and embedding media must fill all spaces within the tissue to support cellular components adequately during microtomy. &ensity of the hardened medium should approach that of the densest tissue component otherwise section deformation will result. The matrix must be elastic enough to recover sectioning deformation, and plastic enough

to facilitate thin sectioning*0. Tissue%medium adhesion is enhanced if the embedding matrix has a fine uniform crystalline morphology which intimately contacts the tissue. Biscosity and melting point of the infiltration medium partly determine the duration and temperature of processing conditions.

$rocessing conditions
Temperature, pressure and agitation reduce the duration of tissue processing and improve the quality of infiltration. TEM$ERATURE 't low temperatures structural elements of tissues are stabilised against the destructive effects of solvent changes*+. This is possibly because of the stiffening and strengthening effect of cold upon biopolymers resulting from diminution in thermal disruption of secondary bonds of the tissue constituents*+. <nfortunately at low temperatures reagent viscosities increase and diffusion rates decrease, resulting in prolonged processing times. 2sothermally processed mammalian tissues show finer detail and less artefacts than those processed by the more practicable, common an%isothermic techniques"7. -eat increases the kinetic energy of molecules and rate of diffusion, with a corresponding decrease in solution viscosity. The application of mild heat within the range *9:. to 1+:., during the dehydration and clearing steps considerably reduces processing times"5,*8, but may concomitantly increase shrinkage(". Tissue shrinkage during infiltration in paraffin wax results mainly from the effect of heat on collagen"8. -igh infiltration temperatures cause marked tissue shrinkage and hardening"9,(" which can be avoided by maintaining embedding waxes (%*:. above their melting points(". $rolonged immersion in paraffin wax at the correct temperature results in only slight tissue shrinkage"8,*9 though tissues such as blood, muscle and yolk may harden and become brittle. The extent to which tissues are affected during paraffin wax infiltration depends upon the combination of fixative, dehydrant and transition solvent used"9,((,(*,*5 as well as the tissue type. icrowave stimulated processing involves complex molecular interactions, the key element of which is internal heating, with stimulation of diffusion*1, and concomitant reduction in the duration of tissue processing. $RESSURE AND .ACUUM -igh pressure facilitates infiltration of dense specimens with viscous resinous embedding media at the block forming stage"7, but is rarely employed for biological specimens. $ositive pressures for fluid transfer that are encountered in closed system processors are probably too low to have a significant influence on tissue infiltration. Bacuum applied during dehydration, clearing and infiltration stages improves the quality of processing. Tissues, particularly lung, are de%aerated, and the solvent boiling point is reduced, thus facilitating evaporation of the reagent from the molten infiltration medium. &uration of wax infiltration is dependent upon viscosity and is not reduced by the application of vacuum*7.

A ITATION >luid interchange between processing reagents and tissues is promoted by exposure of the maximum tissue surface area to reagents. 2f tissues are allowed to settle on the bottom of a container, remain static in the reagent, or are too tightly packed in the processor basket, tissue surface area available for fluid exchange will be restricted and the concentration gradient between the fluid inside and outside the tissues will be low. Ceagent diffusion time is therefore increased and if the duration of processing is not correspondingly increased, inadequate processing will result. &uring processing, tissues should be loosely packed, suspended and agitated within the medium to facilitate the exchange of dilute reagent from the tissues with the more concentrated reagent replacing it. 'gitation of tissues and fluids in manual processing is achieved using rotors or magnetic stirrers. 2n automatic tissue processors, continual rotary or vertical motion of tissue containers, or tidal action and flow of processing fluids ensures adequate fluid exchange. 2deally tissue cassettes should be placed in processors so that the cassette perforations are perpendicular to the fluid flow. >or efficient and effective processing there should be a specimen volume to processing fluid volume ratio of at least "/+0. 'lternate vacuum and positive pressure cycles during processing may provide some micro agitation within tissues, but this has yet to be substantiated. 2n ultrasonic stimulated processing10%1" tissues and fluids are sub@ected to high frequency agitation and associated phenomena, with simultaneous reduction in processing time.

De('dration
The first step in processing is dehydration. Water is present in tissues in free and bound !molecular# forms. Tissues are processed to the embedding medium by removing some or all of the free water. &uring this procedure various cellular components are dissolved by dehydrating fluids. >or example, certain lipids are extracted by anhydrous alcohols, and water soluble proteins are dissolved in the lower aqueous alcohols1(. &ehydration is effected as follows/

&ilution dehydration, the most commonly used method. Specimens are transferred through increasing concentrations of hydrophilic or water miscible fluids which dilute and eventually replace free water in the tissues. .hemical dehydration, where the dehydrant, acidified dimethoxypropane or diethoxypropane, is hydrolysed by free water present in tissues to form acetone and methanol1*%+0 in an endothermic reaction.

&ehydration is necessary in all infiltration methods, except where tissues are simply externally supported by an aqueous embedding medium. .hoice of a dehydrant is determined by the nature of the task, the embedding medium, processing method, and economic factors. &ehydrants differ in their capacity to cause tissue shrinkage !>ig. *#.

2n the paraffin wax method, following any necessary post fixation treatment, dehydration from aqueous fixatives is usually initiated in 803%903 ethanol, progressing through 703%7+3 ethanol, then two or three changes of absolute ethanol before proceeding to the clearing stage. Whilst well fixed tissues can be transferred directly to 7+3 ethanol,+" incompletely fixed tissues may exhibit artefacts if placed directly in higher alcohols. The dehydrant concentration at which processing is initiated depends largely upon the fixative employed. >ollowing fixation in anhydrous fixatives such as .arnoy=s fluid, for example dehydration is initiated in "003 ethanol. To minimise tissue distortion from diffusion currents, delicate specimens are dehydrated in a graded ethanol series from water through "03%(03%+03%7+3%"003 ethanol.*8 &uration of dehydration should be kept to the minimum consistent with the tissues being processed. Tissue blocks " mm thick should receive up to *0 minutes in each alcohol, blocks + mm thick require up to 70 minutes or longer in each change. Tissues may be held and stored indefinitely in 903 ethanol without harm. ;ther dehydrants, including universal solvents, are used in a similar manner to that described for ethanol, though generally in different concentration increments.

De('drating agents
A-CO0O-S These are clear, colourless, flammable, hydrophilic liquids, miscible with water and, when anhydrous, with most organic solvents. 2n addition to their role as dehydrants, alcohols also act as secondary coagulant fixatives during tissue processing. Et(anol is probably the most commonly used dehydrant in histology. 2t is supplied as 77.5+3 ethanol !absolute ethanol, "00 -igh Drade or Standard Drade# and as special ethylated Spirits !77.5+3 ethanol denatured with (3 methanol#. 6oth are satisfactory for histological purposes. ,thyl alcohol formulations differ in standards and nomenclature worldwide and it may be necessary to consult various tables to ascertain the ethanol concentration. ,thanol is a rapid, efficient and widely applicable dehydrant. 2t is normally a poor lipid solvent except under microwave processing conditions*1. ,thanol dissolves nitrocellulose slowly unless combined in equal proportions !or better, "/(#+* with diethyl ether. $rocessing times in absolute ethanol should be minimal. $rogressive removal of bound water from carbohydrates and proteins during prolonged immersion in absolute ethanol causes tissues to harden excessively and become brittle"7,((%(*. .olloid, blood, collagen and yolky tissues are particularly affected"7. The problem is exacerbated by heat during wax infiltration. 'nhydrous cupric sulphate added to the final absolute ethanol on a tissue processor scavenges any water present. The salt is self%indicating/ white when anhydrous, blue when hydrated, and is only slightly soluble in ethanol. 2t is prepared by carefully heating

hydrated cupric sulphate until it turns white, on a hotplate at (+0:.. .ool in a desiccator. 'nhydrous calcium sulphate !&rierite# or molecular sieves act in a similar manner but are non%indicating. Solid drying agents are placed in a "%( cm thick layer in the reagent container, and covered with filter paper or fine sponge to avoid mixing with the specimens during tissue agitation. Met(anol is a good ethanol substitute+1 but rarely used for routine processing because of its volatility, flammability and cost. 2t is a poor lipid solvent, and will not dissolve nitrocellulose unless mixed with acetone. 2n microwave processing it tends to harden tissues more than ethanol*1. Isopropanol was first suggested as an ethanol substitute during the prohibition era in the <nited States+1. 2t is a universal solvent available as 77.53 !absolute# isopropanol, slightly slower in action and not as hygroscopic as ethanol, but a far superior lipid solvent. 2sopropanol is completely miscible with water and most organic solvents, is fully miscible with melted paraffin wax++, and is readily expelled from tissues and wax baths. 2sopropanol shrinks and hardens tissues less than ethanol+1%+9 and is used to dehydrate hard, dense tissues, which can remain in the solvent for extended periods without harm. To minimise shrinkage, fixed tissues are transferred via 803%903 isopropanol or ethanol to absolute isopropanol. 2sopropyl alcohol has also been recommended as a xylene substitute+5. 2n microwave stimulated processing, though unsatisfactory as a dehydrant, isopropanol is used as a transition solvent following ethanol dehydration*1. 2sopropanol only dissolves nitrocellulose in the presence of esters+7 such as methyl benzoate or methyl salicylate, and is used in methyl salicylate%based double%infiltration methods. 2t cannot be used as a dehydrant in alcohol%ether%celloidin techniques. 2sopropanol is a solvent for some lipid%soluble dyes, but is not used in staining work stations as many other dyes are insoluble in this solvent. Normal and tertiar' %utanols are universal solvents mainly used for small%scale manual processing of plant and animal tissues in teaching and research. Aormal butanol is recommended for processing lightly chitinised arthropods*5 and rodent tissues. 2t causes less hardening and shrinkage than ethanol,*5,80 though this is offset by the prolonged processing schedules which may result in tissue shrinkage."5,(( A%butanol is poorly miscible with water and only slowly miscible with paraffin wax. 2t is flammable, with a penetrating camphor%like odour, and the vapours are eye irritants. 2so%butanol, with similar properties and processing characteristics((,(* is a less costly substitute for n% butanol. Tertiary%butanol is widely used in plant histology8" but rarely for animal tissues"9,(*. 6elow (8:. it is hygroscopic crystalline solid, a ma@or disadvantage. 2n processing it is used in a similar manner to n%butanol.8" -/CO-1ET0ERS <nlike the alcohols, these reagents do not act as secondary fixatives, and apart from solvent effects do not appear to alter tissue reactivity.

21Et(o)'et(anol, ethylene glycol monoethyl ether, cellosolve or oxitol is used as a dehydrant preceding polyester wax embedding,"7 for dehydration following dioxane% based fixation of hard animal tissues8(, and in the agar%ester wax double embedding technique. ,thoxyethanol is a colourless, nearly odourless flammable liquid, strongly hygroscopic, miscible with water and most organic solvents. .ellosolve dissolves nitrocellulose and tends to decompose on exposure to sunlight. 2t is rapid but non%hardening in action, and tissues can remain in it for years8(. To avoid severe shrinkage, tissues are transferred from aqueous fixative or washing via 803%903 ethanol into full strength cellosolve. Dio)ane, ",1 diethylene dioxide causes less tissue shrinkage and hardening than ethanol"9,((%(*,8(%8* and is excellent for tissues excessively hardened by ethanol%xylene processing. 2t has a rapid but gentle action, and is best used in a graded series. Tissues may remain in it for long periods without harm. 2t is a colourless, flammable universal solvent with an odour similar to butanol, freezes at "(:., and is miscible with water, most organic solvents and paraffin wax. &ioxane dissolves mercuric chloride, but precipitates potassium dichromate and other salts. 2t is cumulatively toxic and a suspected carcinogen81. &ioxane is expensive and is normally reclaimed by drying over a "0%(0 mm layer of calcium oxide or anhydrous cupric sulphate. .alcium chloride should not be used as it reacts with dioxane and swells8*. &ioxane is also recovered by freezing hydrated solvent in a spark%proofed refrigerator at (%+:.. Water, which separates out, is decanted from the crystalline dioxane which is then thawed, finally dried over a solid dehydrant and reused8+. ,xplosive peroxides form in dioxane exposed to air. They accumulate in recycled solvent which should be periodically tested for the presence of peroxides.88 $ol'et('lene gl'cols !$,D# are water miscible polymers used to dehydrate and embed substances labile to the solvents and heat of the paraffin wax method. They are clear, viscous, slightly hydroscopic liquids or solids of low toxicity. $olyethylene glycols are miscible with most organic solvents and dissolve nitrocellulose. &ehydration is initiated in the low molecular weight liquid glycols. Tissues pass through glycols of increasing molecular weight and viscosity, and are finally embedded in a high molecular weight $,D which is solid at room temperature. $olyethylene glycol used for dehydration can be regenerated by heating at "01:. for (1 hours89. OT0ER DE0/DRANTS Acetone is a colourless flammable liquid with sharp characteristic ketonic odour, low toxicity and is freely miscible with water and organic solvents. 2t is a fast, effective dehydrant though it may cause tissue shrinkage) it may also act as a coagulant secondary fixative. 'cetone is the best dehydrant for processing fatty specimens. Tissues are dehydrated through four changes of acetone, the last of which should always be fresh.

Tissues can be transferred directly from acetone to paraffin wax as the solvent boils off under vacuum85. -owever a transition solvent is normally interposed before the paraffin baths. 'cetone is not recommended for microwave processing as it causes excessive nuclear shrinkage*1. Tetra('drofuran is a colourless, highly volatile and flammable universal solvent with an offensive ethereal odour. 2t is completely miscible with water, most organic solvents, paraffin wax and mounting media. 2t dissolves mountants, but not most dyes. The solvent dehydrates rapidly causing little shrinkage or hardening, and is possibly the best of the universal solvents.87 2t is less toxic than dioxane for which it can be substituted. Tissues are processed as in dioxane method. Tetrahydrofuran can form explosive peroxides which renders solvent recovery distillation dangerous88. 2&2 dimet(o)'propane !& $# and (,( diethoxypropane !&,$# are used for chemical dehydration of tissues.1*%+" They are flammable and form peroxides.+0 & $ and &,$ are miscible with paraffin wax however methanol, one of the hydrolysis products, is not wax miscible and a post dehydration rinse in acetone,15 a transition solvent such as methyl salicylate17 or toluene should precede infiltration with wax. & $ shrinks tissues slightly less than &,$.+" .hemical dehydration is suitable for rapid manual processing or machine processing, and is comparable to conventional dehydration for tissue morphology and staining reactions.+" 'cidified & $4&,$ can be reused several times, though dehydration times need to be extended. The reagent is stored at 1:. in a spark%proofed refrigerator. $(enol, beechwood creosote and aniline facilitate dehydration when mixed with transition solvents, as in Weigert=s carbol%xylol !xylene 9+ ml and phenol (+ ml#.*8 The coupling action permits tissues and celloidin sections to be cleared from lower strength alcohols. .reosote and aniline are used less commonly though in a similar manner to phenol. $henol consists of clear hygroscopic acicular crystals and is also available as 503 w4w liquefied phenol. 2t is soluble in water, alcohol and most organic solvents except petroleum ethers. .oncentrated solutions coagulate nitrocellulose. ;n exposure to air and light, phenol and its solutions develop a pink to reddish discolouration. .ontainers must be protected from light and tightly sealed. $henol crystals and 503 concentrate react violently with formaldehyde.

Clearing
.learing is the transition step between dehydration and infiltration with the embedding medium. any dehydrants are immiscible with paraffin wax, and a solvent !transition solvent, ante medium, or clearant# miscible with both the dehydrant and the embedding medium is used to facilitate the transition between dehydration and infiltration steps. Shrinkage occurs when tissues are transferred from the dehydrant to the transition solvent, and from transition solvent to wax"9,"5 !>ig 1#. 2n the final stage shrinkage may result from the extraction of fat by the transition solvent."5 The term clearing arises because some solvents have high refractive indices !approaching that of dehydrated fixed tissue protein# and, on immersion, anhydrous tissues are rendered transparent or clear.90 This property is used to ascertain the endpoint and

duration of the clearing step. The presence of opaque areas indicates incomplete dehydration. -owever, other solvents, notably chlorinated hydrocarbons, do not render tissues transparent and the clearing endpoint !generally when the specimen sinks in the solvent# must then be determined empirically. Transition solvents extract certain tissue substances such as lipids, but otherwise do not alter tissue reactivity nor behave as secondary fixatives during processing. .hoice of a clearing agent depends upon the following/

the type of tissues to be processed, and the type of processing to be undertaken the processor system to be used intended processing conditions such as temperature, vacuum and pressure safety factors cost and convenience.

Transition sol"ents
00/DROCAR!ONS These are odourless flammable liquids with characteristic petroleum or aromatic odours, miscible with most organic solvents and with paraffin wax. They coagulate nitrocellulose. Toluene and )'lene clear rapidly and tissues are rendered transparent, facilitating clearing endpoint determination. .oncerns over the exposure of personnel to xylene88 relate mainly to the use of the solvent in coverslipping rather than in processing and xylene substitutes can be used in these circumstances. Eylene hardens tissues fixed in non%protein coagulant fixatives and prolonged clearing in the solvent should be avoided. Tissues stabilised in protein coagulant fixatives !6ouin=s or S<S'# are less affected."9,(* 6enzene is more gentle and rapid than xylene and toluene and is probably the best transition solvent, though toxicity and possible carcinogenicity81 preclude its use in histology. 2ndustrial grade xylene may contain nearly (+3 of other solvents such as ethyl benzene, with traces of benzene, odorous mercaptans and hydrogen sulphide. ;nly the sulphur and benzene%free solvent%grade xylene should be used for histological purposes. $etroleum sol"ents have a gentle, non%hardening action on tissues, clear more slowly than xylene and toluene, and do not render tissues transparent. 6lends of aromatic, naphthenic and aliphatic solvents !each with varying toxicity, flammability and solvent action# can be used as xylene substitutes.88 any of these solvents have a particularly strong petroleum odour which some people find ob@ectionable. Toxic effects of petroleum solvents are broadly similar to those of pure hydrocarbons % skin degreasing, acute intoxication and narcosis in high concentrations. 6lends with high aromatic and naphthene but reduced paraffin content such as Shell E*69", are good, moderately toxic, high flash%point solvents. Those with high paraffin but little or no naphthene and aromatic content often have low flammability and toxicity, and a slow and gentle clearing

action. Ferosene, some xylene substitutes and Shellsol "8(8 have properties intermediate between these two groups. C(lorinated ('drocar%ons are colourless solvents with sweet odours and are miscible with most organic solvents and with paraffin wax. They are good lipid solvents and do not dissolve nitrocellulose or render tissues transparent. embers of this group clear more slowly but harden far less than xylene. 'lthough non%flammable, solvents in this group decompose in the presence of heat to form phosgene and hydrochloric acid. They are all narcotic and toxic to varying degrees. .hlorinated hydrocarbons are ozone destroying chemicals, and from Ganuary "778 ","," trichloroethane and carbon tetrachloride are banned from use under the ontreal $rotocol.9( C(loroform is an expensive, heavy, highly volatile, slowly penetrating transition solvent. 2t causes less brittleness than xylene and is often used on dense tissues such as uterus and muscle*( which can be cleared overnight without undue hardening. Since chloroform attacks some plastics and sealants its use may be restricted in certain closed system processors. Car%on tetrac(loride has similar properties, but because of its high toxicity is now rarely used in histology. Tric(loroet(ane and other members of this group are commonly used as xylene9*,91 and chloroform substitutes. They include ","," trichloroethane !","," T.,#, present in 2nhibisol) ","," T., and perchloroethylene components of .A$*0 and -istosol) and trichloroethylene. These solvents are stable to light but tend to slowly liberate hydrochloric acid on contact with water. They contain stabilisers to inhibit reactions with aluminium and its alloys.+7 'lthough mildly toxic !except at high concentrations#81 the decision to substitute them for more toxic solvents must be soundly based !Table 9.*#9+. 6ecause of their high volatility, members of this group may achieve and exceed maximum allowable concentrations in poorly ventilated laboratories far more rapidly than xylene under the same conditions.9+ ESTERS These are colourless flammable solvents miscible with most organic solvents and with paraffin wax. n1!ut'l acetate is used as a xylene substitute98 and nitrocellulose solvent. Am'l acetate, met('l %en3oate and met('l salic'late are chiefly used as nitrocellulose solvents in double embedding techniques. They have low toxicity, but their strong penetrating odours necessitate good laboratory ventilation. They are ideal for manual processing as tissues may be left in them for extended periods without hardening. These esters are difficult to eliminate from paraffin wax and should be extracted from tissues with one or two brief changes of toluene or similar solvent before passing through two or three changes of wax. ethyl benzoate and methyl salicylate render tissues completely transparent and are used for clearing helminth parasites for examination and whole

mounting. ethyl salicylate clears tissues from 783 ethanol, hardens less and has a more pleasant odour than methyl benzoate. 2t causes minimal tissue shrinkage and hardening"5 and tissues can remain in it indefinitely without harm. This ester is one of the best though expensive transition solvents. TER$ENES Terpenes are isoprene polymers found in essential oils originally derived from plants,99 though some are now synthesised. They are the earliest transition solvents to be used in histology95 and include turpentine and oils of bergamot, cedarwood, clove, lemon, origanum and sandalwood.8",97 2n general the natural oils are not highly pure compounds but contain several substances. any terpenes clear tissues and celloidin sections from 503%7+3 alcohol, render tissues transparent and have a slow gentle non%hardening action. ost are generally regarded as safe though some have particularly strong odours which can be overpowering, requiring good laboratory ventilation. When used for processing hard, dense or chitinised non%mammalian tissues, terpenes may need to be diluted with the anhydrous dehydrant and with wax in a series, with terpene/dehydrant or wax ratios of */", "/", "/* followed by three or four changes of pure wax. Tissue penetration is aided and shrinkage minimised by diluting viscous terpenes. Terpenes have low evaporation rates and are difficult to eliminate from paraffin wax, necessitating one or two *0 minute changes of toluene or similar solvent to remove the terpene before infiltration with wax. 6rief immersion in toluene does not negate the effectiveness of the terpene. 'lternatively, tissues are given three, four or more changes of wax until the terpene has been eliminated. 'lthough biodegradable, terpenes are not water miscible and should not be flushed away with water, but disposed of by recycling or incineration. Cedar4ood oil& largely composed of cedrene, rapidly clears tissues from 7+3 alcohol, hardens tissues the least of all the transition solvents, but is difficult to eliminate from tissues during wax infiltration. 2t is particularly useful for processing dense tissues such as uterus or scirrhous carcinomas, and has a role in forensic histopathology in processing the hardened skin margins of electrical burns and bullet wounds. Tissues can remain in cedarwood oil indefinitely without harm. ?ow viscosity refined oil should be used for clearing. >ormation of crystalline cedrol in cedarwood oil can be overcome by the addition of " ml xylene or toluene to 50 ml cedarwood oil8+. .edarwood oil is expensive, but exhausted oil can be restored by filtering, then heating to 80:. under vacuum for *0% 80 minutes50. -imonene !dH limonene# is derived from citrus fruit and is a component of various proprietary blends of transition solvents such as .learene, -emo%&e and -isto%.lear marketed as xylene substitutes. 2t is less viscous than cedarwood oil and is similar to the esters in clearing action and in elimination from wax. ?imonene may cause allergic skin reactions.5",5(

Terpineol is a clear almost colourless mixture of isomers with a faint pleasant odour and very low evaporation rate. 2t clears tissues from 503%703 alcohol with minimal hardening. ?ike the other terpenes it is difficult to eliminate from paraffin wax. 2t is a particularly useful substitute for cedarwood oil in manual processing and is also used in open%dish microscopic examination of cleared parasitic helminths. Tissues may remain in it indefinitely without harm. Some of the specific safety requirements relating to processing solvents are summarised in Table *.

Infiltration and em%edding media and met(ods

2deally an infiltrating and embedding medium should be/"7

soluble in processing fluids suitable for sectioning and ribboning molten between *0:. and 80:. translucent or transparent) colourless stable homogeneous capable of flattening after ribboning non%toxic odourless easy to handle inexpensive

2n addition the properties of the medium should approach those of the tissues to be sectioned with regard to density, elasticity, plasticity, viscosity and adhesion and should be harmless to the embedded material. Barious substances have been used to infiltrate and embed tissues for microtomy. Aone completely fulfil the foregoing criteria, and media are selected according to the nature of the task for which they are required. Em%edding is the process by which tissues are surrounded by a medium such as agar, gelatine, or wax which when solidified will provide sufficient external support during sectioning. Infiltration is the saturation of tissue cavities and cells by a supporting substance which is generally, but not always, the medium in which they are finally embedded. Tissues are infiltrated by immersion in a substance such as a wax, which is fluid when hot and solid when cold. 'lternatively, tissues can be infiltrated with a solution of a substance dissolved in a solvent, for example nitrocellulose in alcohol%ether, which solidifies on evaporation of the solvent to provide a firm mass suitable for sectioning.

Dou%le em%edding is the process by which tissues are first embedded or fully infiltrated with a supporting medium such as agar or nitrocellulose, then infiltrated a second time with wax in which they are also embedded. In"estment generally refers to the practice of embedding wax infiltrated tissues in another wax, such as $iccolyte%paraffin wax, modified to provide improved tissue support and sectioning qualities. .acuum infiltration is the impregnation of tissues by a molten medium under reduced pressure. The procedure assists the complete and rapid impregnation of tissues with wax, reduces the time tissues are sub@ected to high temperatures thus minimising heat%induced tissue hardening, facilitates complete removal of transition solvents, and prolongs the life of wax by reducing solvent contamination. Bacuum infiltration requires a vacuum infiltrator or embedding oven, consisting of wax baths, fluid trap and vacuum gauge, to which a vacuum of up to 980 mm -g is applied using a water or mechanical pump. odern tissue processors are equipped to deliver vacuum, or vacuum and pressure, to all or some reagent stations during the processing cycle.

$arffin 4a)
$RO$ERTIES $araffin wax is a polycrystalline mixture of solid hydrocarbons produced during the refining of coal and mineral oils. 2t is about two thirds the density and slightly more elastic than dried protein.** Wax hardness !viscosity# depends upon the molecular weight of the components and the ambient temperature. -igh molecular weight mixtures melt at higher temperatures than waxes comprised of lower molecular weight fractions. $araffin wax is traditionally marketed by its melting points which range from *7:. to 85:.. Tissue%wax adhesion depends upon crystal morphology of the embedding medium. Small, uniform sized crystals provide better physical support for specimens through close packing. .rystalline morphology of paraffin wax can be altered by incorporating additives which result in a less brittle, more homogeneous wax with good cutting characteristics. There is consequently less deformation during thin sectioning. Setting temperature does not appreciably affect crystal size.51%58 MODI*IED $ARA**IN 5A,ES The properties of paraffin wax are improved for histological purposes by the inclusion of substances added alone or in combination to the wax/

improve ribboning/ prolong heating of paraffin wax at high temperatures or use micro%crystalline wax increase hardness/ add stearic acid decrease melting point/ add spermaceti or phenanthrene improve adhesion between specimen and wax !alter crystalline morphology#/ add 0.+3 ceresin, 0."%+3 beeswax, rubber, asphalt, bayberry wax, or phenanthrene.59

,arly histological wax formulations*8,55%57 have largely been replaced by uniform, high quality proprietary blends of histological paraffin waxes. 'dditives recently incorporated in proprietary waxes include the following/ $iccol'te 667& a thermoplastic terpene resin added at the rate of +3%"03 to the infiltrating wax,70 or to the final investing paraffin wax to improve tissue support for thin sectioning and facilitate flattening and expansion of sections on the waterbath.7"%7( $iccolyte mixtures cannot be used in certain models of fluid%transfer type tissue processors. $lastic pol'mers such as polyethylene wax, added to improve adhesion, hardness and plasticity.7* $olymer waxes are incorporated in the ma@ority of proprietary histological paraffin wax blends presently available. Dimet('l sulp(o)ide !& S;# added to proprietary blends of plastic polymer paraffin waxes reduces infiltration times and facilitates thin sectioning.71 & S; scavenges residual transition solvent and probably alters tissue permeability by substituting for or removing bound water thus improving infiltration. Some individuals who handle & S;% paraffin wax may experience an unpleasant and annoying oyster or garlic taste probably caused by & S; metabolites.7+ $ossible health risks associated with the use of & S;% paraffin wax88 are minimal if correct laboratory hygiene is practised.

Em%edding tissues in paraffin 4a)

Tissues are embedded by placing them in a mould filled with molten embedding medium which is then allowed to solidify. ,mbedding requirements and procedures are essentially the same for all waxes, and only the technique for paraffin wax is provided here in detail. 't the completion of processing, tissues are held in clean paraffin wax which is free of solvent and particulate matter. Cequirements for embedding are as follows/

a supply of clean, filtered paraffin wax held at (%1:. above its melting point. a cold plate to rapidly cool the wax. a supply of moulds in which to embed the tissues.

These elements are conveniently combined in commercially available embedding stations !>ig +#. ;therwise a wax dispenser, embedding oven and ice will suffice. There are four main mould systems and associated embedding protocols presently in use78 !>ig 8#/ traditional methods using paper boats) ?euckart or &immock embedding irons or metal containers) the $eel%a%way system using disposable plastic moulds and) systems using embedding rings or cassette%bases which become an integral part of the block and serve as the block holder in the microtome. eneral Em%edding $rocedure
MET0OD

" ;pen the tissue cassette, check against worksheet entry to ensure the correct number of tissue pieces are present. ( Select the mould, there should be sufficient room for the tissue with allowance for at least a ( mm surrounding margin of wax.

* >ill the mould with paraffin wax. 1 <sing warm forceps select the tissue, taking care that it does not cool in the air) at the same time. + .hill the mould on the cold plate, orienting the tissue and firming it into the wax with warmed forceps. This ensures that the correct orientation is maintained and the tissue surface to be sectioned is kept flat. 8 2nsert the identifying label or place the labelled embedding ring or cassette base onto the mould. 9 .ool the block on the cold plate, or carefully submerge it under water when a thin skin has formed over the wax surface. 5 Cemove the block from the mould. 7 .ross check block, label and worksheet. ORIENTATION O* TISSUE IN T0E !-OCK .orrect orientation of tissue in a mould is the most important step in embedding. 2ncorrect placement of tissues may result in diagnostically important tissue elements being missed or damaged during microtomy. 2n circumstances where precise orientation is essential tissue should be marked or agar double embedded. <sually tissues are embedded with the surface to be cut facing down in the mould. Some general considerations !>ig.9# are as follows/

elongate tissues are placed diagonally across the block tubular and walled specimens such as vas deferens, cysts and gastrointestinal tissues are embedded so as to provide transverse sections showing all tissue layers tissues with an epithelial surface such as skin, are embedded to provide sections in a plane at right angles to the surface !hairy or keratinised epithelia are oriented to face the knife diagonally# multiple tissue pieces are aligned across the long axis of the mould, and not placed at random.

&uring cooling, paraffin wax shrinks up to "+3, causing compression in tissues."9 This compression is almost fully recovered when sections are floated on a warm waterbath8) compression resulting from microtomy is only partially recovered.

$rocessing met(ods and routine sc(edules

Tissues are usually more rapidly processed by machine than by manual methods, although the latter can be accelerated by using microwave or ultrasonic stimulation. >or routine purposes tissues are most conveniently processed through dehydration, clearing and infiltration stages automatically by machine. There are two broad types of automatic tissue processors % tissue%transfer and fluid%transfer types.

Automated tissue processing

TISSUE1TRANS*ER $ROCESSORS These processors are characterised by the transfer of tissues, contained within a basket, through a series of stationary reagents arranged in%line or in a circular carousel plan. The rotary or carousel is the most common model of automatic tissue processor, and was

invented by 'rendt in "707.97 2t is provided with 7%"0 reagent and (%* wax positions, with a capacity of *0%""0 cassettes depending upon the model. >luid agitation is achieved by vertical oscillation or rotary motion of the tissue basket. $rocessing schedules !Table 1# are card%notched, pin or touch pad programmed. Tissue%transfer processors allow maximum flexibility in the choice of reagents and schedules that can be run on them, in particular, metal%corrosive fixatives, a wide range of solvents, and relatively viscous nitrocellulose solutions can all be accommodated. These machines have a rapid turn%around time for day4night processing. 2n more recent models !>ig.5# the tissue basket is enclosed within an integrated fume hood during agitation and transfer cycles thus overcoming the disadvantages of earlier styles. *-UID1TRANS*ER $ROCESSORS 2n fluid%transfer units !>ig.7# processing fluids are pumped to and from a retort in which the tissues remain stationary. There are "0%"( reagent stations with temperatures ad@ustable between *0%1+:., *%1 paraffin wax stations with variable temperature settings between 15%85:., and vacuum%pressure options for each station. &epending upon the model these machines can process "00%*00 cassettes at any one time. 'gitation is achieved by tidal action. Schedules are microprocessor programmed and controlled. Bacuum%pressure cycles coupled with heated reagents allow effective reductions in processing times and improved infiltration of dense tissues. >luid%transfer processors overcome the main drawbacks of the tissue%transfer machines. Tissues are unable to dry out within the sealed retort and reagent vapours are vented through filters or retained in a closed%loop system. $rocessors are provided with alert systems and diagnostic programs for troubleshooting and maintenance. Some models are unable to accept mercury or dichromate%based fixatives, certain solvents, for example chloroform, or wax additives such as $iccolyte. Cepresentative schedules for rapid and overnight processing are provided in Table +. TISSUE RECO.ER/ $ROCEDURES $rocedures for recovery of tissues that have air dried because of mechanical or electrical failure of the processor are similar to those used for mummified specimens. Tissues accidentally returned into fixative or alcohol following wax infiltration are recovered by methods outlined in Table 8. ENERA- CONSIDERATIONS 6askets and metal cassettes should be clean and wax%free. Tissues should not be packed too tightly in baskets so as to impede fluid exchange. $rocessors must be free of spilt fluids and wax accumulations to reduce hazards and to ensure mechanical reliability. >luid levels must be higher than the specimen containers. Timing and delay mechanism must be correctly set and checked against the appropriate processing schedule. ' processor log should be kept in which the number of specimens processed, processing reagent changes, temperature checks on the wax baths and the completion of the routine

maintenance schedule, is recorded as an integral part of the laboratory quality assurance program.

Manual tissue processing

anual tissue processing is usually undertaken for the following reasons/

power failure or breakdown of a tissue processor a requirement for a non%standard processing schedule as for/ o rapid processing of an urgent specimen o delicate material o very large or thick tissue blocks o hard, dense tissues !nitrocellulose methods# o special diagnostic, teaching or research applications small scale processing requirements resin embedding.

The main advantage of manual processing over automated methods lies in the flexibility of reagent selection, conditions and schedule design to provide optimum processing for small batches of tissues. ,xposure of tissues to the deleterious effects of some reagents can be carefully monitored and regulated through observation and precise timing. There is usually considerable latitude in the processing times given in schedules although maximum rather than minimum times should be used, as it is better to extend processing rather than risk the problems of under processed tissues. anual processing is accelerated using microwave ovens or ultrasonics. Schedules for rapid manual processing using chemical dehydration !Table 9#, one and two day routine processing !Table 5#, and extended manual processing for large, thick, or hard tissues !Table 7# are provided. <niversal solvents with particularly favourable attributes, normally precluded from routine machine processing because of budgetary or safety constraints, can be successfully used in small volumes under controlled conditions for manual processing. Schedules are provided for manual processing using n%butanol !Table "0# and dioxane !Table ""#. Aonetheless manual processing can be time consuming and inconvenient. .are must be exercised so that tissues are left overnight in reagents that will cause minimal harm. ' permanent series of solutions in wash bottles simplifies processing small single specimens. Tissues are processed in tubes and agitated on a rotor. Ceagents are pipetted, or decanted through a fine sieve. ultiple specimens or large blocks are economically processed in large lidded @ars of processing fluids. The specimen to reagent volume ratio should be at least "/+0. 'gitation is provided by a magnetic%stirrer. &ehydrated tissues float on the surface when transferred to higher density transition solvents such as chloroform or cedarwood oil. -owever, if placed in lower density mixtures of dehydrant%transition solvent before finally transferring to pure transition

solvent, tissues will remain submerged throughout the clearing stage. 'n alternative approach is to carefully layer the dehydrant onto the transition solvent and introduce the tissue into the upper layer. The tissue sinks as the dehydrant gradually replaces the transition solvent. Ceagents are carefully decanted and the specimen placed in a fresh change of transition solvent.

Micro4a"e1stimulated processing
Capid manual microwave%stimulated paraffin wax processing of small batches of tissues gives excellent results which are comparable to tissues processed by longer automated non%microwave methods.*1,79%"0" $rocessing is undertaken in a dedicated microwave oven !>ig. "0# which is fitted with precise temperature control and timer, and an interlocked fume extraction system to preclude accidental solvent vapour ignition. 'gitation is provided by an air%nitrogen system. &omestic microwave ovens with a temperature probe and timer accurate to seconds are suitable for tissue processing. ' turntable or in%built radiation disperser facilitates even reagent heating. Toxic and flammable solvent vapours generated during processing cannot always be adequately vented from these ovens and present an ignition hazard if the electrical system is unprotected. ;vens should therefore be used within a fume cupboard to minimise this problem. .alibration of domestic ovens is essential for optimum results and the accuracy of the temperature probe, duration of cycle time, and net power levels at various settings must be determined before the oven is used to process tissues.*1 E8UI$MENT Tissues are processed in conventional plastic cassettes, including those with !provided the metal lids lie below the fluid#.7 Transparent glass or solvent%resistant plastic containers of about (00 ml capacity are ideal for processing batches of up to "1 cassettes per container. *I,ATION >or rapid processing, tissues are fixed by microwave irradiation,7 or in 7+3 ethanol !800 ml#%polyethylene glycol $,D 100 !1+ ml#"0( from which specimens can be transferred directly to dehydrant. >ormaldehyde%fixed tissues must be rinsed in running tap water for + minutes before microwave processing and an extra dehydration change incorporated in the schedule. $rocessing times for formaldehyde%fixed tissues need to be increased above those provided for coagulant%fixed tissues.*1 $icric acid fixed tissues should not be microwave processed as there is an explosion risk even in well washed tissues.*1 $rocessing schedules are provided in Table "(. 0INTS *OR MICRO5A.E $ROCESSIN % Tissue blocks should be as thin as possible. ?ength and width are not as important.*1

$rocess blocks of similar thickness together. Ceagent volumes should be at least +0 times that of specimen volume.

The temperature probe should be placed centrally in processing baths. <se a dummy load to check heat generation should reagents boil on minimum settings % an equal volume of reagent irradiated together with the primary load effectively halves the energy received by the primary load. $re%heat paraffin wax baths in a conventional oven. 'n increase in the number of cassettes or fluid volumes will require a concomitant increase in power and or time to achieve the correct processing temperature.

Ultrasound1stimulated processing
<ltrasonics are used in histopathology to accelerate fixation, tissue processing for electron microscopy, the decalcification of bone, tissue softening in post%embedding ad@uvants, improving the sensitivity of immunohistochemical reactions, conventional staining and for accelerated tissue processing. <nfixed tissue blocks "%( mm thick can be fixed and processed to paraffin wax using ultrasonic%stimulation in " hour 1+ minutes.10%1" The most important effect of ultrasound at frequencies of "00 k-z%" -z is agitation. 't lower frequencies cavitation phenomena and attendant heat, pressure and streaming effects may damage tissues and care must be exercised. $rocessing is performed in reagent containers suspended in a detergent solution within the transducer tank of an ultrasonic cleaner operated at +0 watts. 'n immersion heater is used to elevate bath temperatures for paraffin wax infiltration. Tissues are placed in metal or plastic cassettes for processing. .oagulant fixatives provide optimal stabilisation for ultrasonic%stimulated processing.10 Tissues are dehydrated in ethanol and cleared in toluene, or preferably methyl benzoate or methyl salicylate !Table "*#. .ells and organelles such as cilia, microvilli and desmosomes are all well preserved. ;ld and friable specimens sometimes exhibit marginal distortion and erosion.

Alternati"e em%edding media and processing met(ods

'lternative embedding media may provide optimum support for tissues in applications for which paraffin waxes are unsuited, for example when/

tissue components are heat or reagent labile hard or dense tissues are inadequately supported adhesion between specimen and wax is poor very thick or very thin sections are required sectioning whole organs such as lung or brain.

Cesin embedding methods are now used for many of these applications. Aon%resinous embedding media include those listed below. A8UEOUS MEDIA Agar has a high melting point and low gelling temperature of agar make it ideal for

double embedding multiple small tissue fragments. 'gar is generally unstained by overnight stains, but will stain with alcian blue. elatine is used for simple embedding in a similar manner to agar. -owever the low melting point of gelatine !*+%10:.# makes it unsuitable for double embedding. 2t is used in Dough and Wentworth=s whole%organ sectioning method and its variants, or simply to support large tissue blocks for " mm thick sectioning and subsequent three%dimensional reconstruction."0* 2n phospholipid and enzyme studies tissues may be infiltrated and embedded in gelatine+",90 and the resulting blocks sectioned on a freezing microtome. This technique has now largely been superseded by other media used for cryotomy. Sodium car%o)' met('l cellulose !. .# used as an embedding medium for whole body sectioning techniques, was first developed for autoradiograph studies"01 and subsequently refined for histological and histochemical applications."0+ >rozen tissues are transferred from coolant directly into +3 . ., briefly placed under vacuum to remove trapped air, then frozen to a solid block for sectioning. $ol'"in'l alco(ol !$B'# is a highly polar, water soluble medium suited to a variety of applications, in particular histochemical studies of lipids and enzymes."08%"09 Tissues are infiltrated at elevated or room temperatures through an ascending series of aqueous $B'% glycerol solutions and embedded in "+3 aqueous $B' which is slowly dried to produce a firm block. Sections are cut at "%"00 Im in the normal manner. -umidity control during microtomy is important. $rocessing schedules take "%( weeks or longer, restricting $B' to research applications. ' low molecular weight, low viscosity $B' is necessary for this method."09 Cenewed interest in $B' as an infiltrating and embedding medium for electron microscopy"05 has resulted in refinements to the technique. .ross%linking $B' with glutaraldehyde provides a final hydrophobic block containing some "03 water, with improved sectioning characteristics and good preservation of lipids, proteins and carbohydrates. Tissues are infiltrated through aqueous $B' solutions at concentrations from "3%(+3. $B' of molecular weight "1k&a, 773 hydrolysed gives the most consistent results"05, but must be fully hydrolysed with sodium hydroxide before use. The application of $B' to immunohistochemical studies is worthy of attention. 5ATER1MISCI!-E MEDIA $ol'et('lene gl'cols !$,D# are water soluble media used for investigation of heat and solvent%labile lipids and proteins"07%""0, and to overcome tissue shrinkage, damage and distortion inherent in the paraffin wax technique. The polyethylene glycols, or .arbowaxes, are polymers of varying length !the numerical suffix denotes molecular weight#. 't room temperature $,D (00 and $,D 800 are syrupy liquids, $,D "000 is soft and slippery, $,D "+00 is hard, and $,D 1000 is hard and brittle. 2n general they are less elastic, denser and somewhat harder than paraffin wax. .rystal slip is a bigger problem than in paraffin and sectioning deformation is mainly non%recoverable.** Tissues are dehydrated by gradual infiltration through increasing concentrations of aqueous $,D solutions, to pure $,D in which they are finally embedded. Sections are cut in a low humidity environment, otherwise considerable difficulties arise. They are difficult to flatten without loss of tissue and adhere poorly to slides, leading to the development of

numerous flotation fluids.""0 ?ow viscosity nitrocellulose !?BA#89 or water insoluble polyvinyl acetate resin""0 incorporated into $,D dehydrating, infiltrating and embedding solutions allow water flotation of sections. The $,D dissolves, leaving the tissue in a thin film of ?BA or $B' which is mounted on albumenised slides in the usual manner. These approaches !Table "1# surmount many of the previous problems inherent with $,D. The nitrocellulose is removed from sections by immersing in $,D (00 for "+ minutes. $ol'et('lene l'col1-.N Solutions89 REA ENTS RE8UIRED " $olyethylene glycol 7+ g ( ?BA -E *%+ or *0%+ +%"0 g The ?BA must be finely divided and dried to facilitate dissolution in the $,D media) +3 ?BA is preferred as "03 ?BA is near saturation in $,D. MET0OD The mixtures are heated to 80:. and stirred to aid solution. $roblems with this method include the high viscosity of infiltrating media necessitating slow agitation and uneven distribution of ?BA in the final embedding mix which results in crazed blocks. These can be overcome mostly by thorough blending of the ?BA and $,D. With current interest in immunohistochemistry, polyethylene glycols may warrant re%evaluation. -owever considerable time and patience are required when using these waxes. $ol'et('lene gl'col monostearate !Aonex 8*6#, a water soluble synthetic wax is used in a similar manner to polyethylene glycol and polyester waxes with application in histochemistry""" and botanical histology.""( 5ATER1TO-ERANT MEDIA Diet('lene gl'col distearate is a hard, brittle, water tolerant ester !m.p. 19%+(:.#. 2t has certain deficiencies when used for routine embedding,""* unless combined with other substances as in ester waxes. -owever it may be used unmodified for thin sectioning !0.+%( Im# of freeze dried and osmium tetroxide fixed tissues for high resolution light microscopy.""1%""+ Tissues are dehydrated and cleared as in the paraffin wax method. Ester 4a)es, developed by Steedman,""8 and subsequently modified""9%""5 have low melting points, are hard at room temperature and have good adhesive properties. They are therefore ideal for supporting and serially sectioning refractory hard, chitinised material such as arthropods, and tissues which heat%harden excessively. They are also used for simple investment of paraffin blocks to be sectioned under hot conditions""7 and in double embedding with agar. ,ster waxes are no longer commercially available and must be prepared from the basic ingredients. Ester 5a) 69:;""5 !m.p. 15:.# REA ENTS RE8UIRED " &iethylene glycol distearate 80 g

( Dlyceryl monostearate *0 g * *00 polyethylene glycol distearate "0 g MET0OD " elt the diethylene glycol distearate and heat it until clear. 'dd the glyceryl monostearate and when dissolved add the polyethylene glycol distearate. ( >ilter through coarse filter paper supported in a ring rather than a filter funnel. 2n Tropical ,ster wax "780""7 !m.p. +0:.# triethylene glycol monostearate "0 g is substituted in the forgoing formula for the *00 polyethylene glycol distearate. This modification permits sectioning and ribboning at room temperatures of *9.+:.. Tissues are infiltrated at +8%80:.. ,ster waxes are soluble in 7+3 ethanol, n%butanol, cellosolve and xylene. Tissues are usually dehydrated via (%ethoxyethanol in which the waxes are soluble, obviating the need for a transition solvent. ,ster waxes do not charge with static electricity, and have good ribboning, thin sectioning and glass adhesion properties. $ol'ester 4a), developed by Steedman"(0 is a ribboning, low melting point wax which reduces heat%induced artefacts. 2t is recommended for heat labile tissues,"(" to minimise heat%induced hardening in difficult tissues and is an ideal medium for combined light and scanning electron microscopy of animal tissues."(( The properties of the wax facilitate immunohistochemical investigations as antigenic determinants are well preserved."(* The main advantages of this medium are low melting point and infiltration directly from 783 ethanol permitting a near isothermic processing schedule for mammalian tissues !Table "+#. $olyester wax is no longer commercially available and must be prepared from the basic ingredients. $ol'ester 5a) 9;<6; !m.p. *5:.# REA ENTS RE8UIRED " 100 polyethylene glycol distearate 70 g ( "%hexadecanol !cetyl alcohol# "0 g MET0OD " elt the polyethylene glycol at 80 ., then mix in the cetyl alcohol. ( .ool to a solid from which working quantities are obtained. The wax has good water tolerance and is soluble in most histological dehydrants and transition solvents. 6ecause of its low melting point, non%volatile transition solvents such as cedarwood oil and other terpenes should not be used. $olyester wax adheres to metal embedding moulds and paper%boat or plastic peel%a%way moulds are recommended. Aormally blocks are cut in cool room temperatures using a cooled knife. $olyester wax is more conveniently sectioned on a cryotome at %+:. to ((:.. Sections are affixed to gelatine subbed or aminoalkylsilane treated slides, or floated on amylopectin."(0 6locks and sections are stored at 1:.. $olyester wax is dissolved from sections with absolute ethanol. Steedman"7 proposed simultaneous fixation and infiltration of tissues with picro%

formal%polyester wax and mercury%formal%polyester wax mixtures. These methods and principles present possibilities for immunohistochemistry, but appear to have received little attention. 0/DRO$0O!IC MEDIA Nitrocellulose .elloidin !.# and ?ow Biscosity Aitrocellulose !?BA#, mixtures of di% and tri% nitrocellulose, are composed of yellowish%white matted filaments with the appearance of raw cotton. Aitrocellulose is insoluble in water, but soluble in absolute ethanol%diethyl ether, amyl acetate, methyl benzoate, methyl salicylate and (%ethoxyethanol and is set by most hydrocarbon solvents. 2t is highly flammable, and must be kept alcohol%damped with n%butanol or as 53 solutions in ethanol%ether or "3 ?BA. in methyl benzoate as it is explosive if detonated when dry. .elloidin solutions have a low tolerance of water and dehydration must be thorough. ?BA tolerates up to 83 of water,"(1 has superior penetration and final block hardness and is supplied in various grades of viscosity and nitrogen content.+*,89 Aitrocellulose tissue processing techniques are generally employed for sectioning hard tissues such as bone,"(+%"(8 for topographical studies of central nervous system tissues,"(9 or for delicate embryonic material. Tissues are processed at room temperature producing minimal and shrinkage and hardening. 2mmunohistochemical investigations such as immunophenotyping of lymphoid and non%lymphoid cells"(5 are possible on nitrocellulose processed tissues.

Dou%le em%edding and dou%le infiltration met(ods

&ouble embedding methods such as agar%paraffin embedding, are used when tissues require external support or particular pre%embedment orientation. $araffin wax double infiltration methods provide hard tissues with additional support provided by substances such as agar or nitrocellulose, with the convenience and ease of wax microtomy. A AR1$ARA**IN 5A, DOU!-E EM!EDDIN &ouble embedding in agar%paraffin is a reliable and convenient method of handling minute and friable tissue fragments such as curettings and endoscopic biopsies, which can be lost during tissue processing."(7%"*+ 2t also overcomes the difficulty of manipulating small tissue fragments during embedding and facilitates correct orientation and identification of tissues for histochemical and immunohistochemical control tissues."*8 Agar Em%edding Medium REA ENTS RE8UIRED " 'gar !technical or microbiological grade# ".+ % *.0 g ( &istilled water 70 ml * *93 formaldehyde solution "0 ml MET0OD " &issolve the agar in the distilled water using a boiling water bath, autoclave or microwave oven. ( 'dd formaldehyde and mix well. * &istribute (0 ml aliquot=s into screw capped bottles. Store at 1:..

1 >or use melt the agar as previously indicated, cool to +0%80:., and hold in a 80:. oven. ?oosen container caps before remelting agar. + ,mbed tissues by pipetting agar solution over tissue fragments correctly oriented on a clean flat surface"(7 or membrane filter."0+ TEC0NICA- NOTES " Specimens can be embedded in silicon%rubber flat embedment moulds, moulds made from cut%down plastic syringes,"*0 or in metal Tissue%Tek type moulds."*(,"*+ ( <nstabilized agar embedments sometimes disintegrate during processing and must be stabilised by the addition of (.+3%13 formaldehyde to the medium, or by immersing blocks in 903 ethanol or fixative for "%* hours. Agar1$araffin 5a) Dou%le Em%edding *or *ragments& !iopsies And *ria%le Specimens"*+ REA ENT RE8UIRED 'gar !prepared as above# MET0OD " >ill the appropriate sized Tissue%Tek base mould with agar medium cooled to approximately 10:.. ( Aumber embedding cassette, cross checked with request slip and specimen container. * $lace specimen in agar%filled mould and orientate. 1 'llow agar to solidify for + minutes. $lace embedding cassette on top of the mould to identify the tissue. + When the agar block is solid, detach the specimen from the mould by sliding a scalpel down one side. Trim and notch the agar block as required, leaving a *%+ mm width of agar surrounding the tissue. $rocess normally. Agar1$araffin 5a) Dou%le Em%edding *or !one Marro4 Aspirates And Cell Suspensions Using T(e Collodion !ag Tec(ni+ue"*9 REA ENT RE8UIRED .ollodion !add 13 nitrocellulose to (+ ml absolute ethanol / 9+ ml diethyl ether# MET0OD " $lace "%( ml of collodion into a "0 ml glass centrifuge tube. Cotate the tube so that an even layer of collodion coats and sets on the inside surface) gentle blowing into the tube hastens the setting process. ( >ill the tube with 903 ethanol and stand for + minutes to harden the collodion. &rain the ethanol, rinse with water. * 'dd fixed aspirate or suspension to tube. 1 .entrifuge at (000 rpm for *0 seconds. + &ecant supernatant fluid. 8 $ipette "%( ml of agar, cooled to approximately 1+:., into the centrifuge tube. 9 Capidly resuspend the specimen and centrifuge at (000 rpm for " minute. 5 'llow the mass to cool for + minutes. 7 <sing fine forceps, carefully detach the collodion bag and contents from the tube.

"0 Trim off unwanted collodion, and process as a normal specimen, taking care to orient and embed the specimen correctly. A AR 1 ESTER 5A, DOU!-E IN*I-TRATION &ouble infiltration of tissues in agar and ester wax"*5%"*7 aids thin serial sectioning of chitinised tissues at 0.+%".0 Im and is a possible alternative to pine resin%beeswax paraffin wax used to support plastic vascular prostheses for sectioning."10 The fine crystalline nature and hardness of ester wax improves tissue%wax adhesion and provides adequate support for thin serial sectioning. Agar1Ester 5a) Dou%le Infiltration"*5 REA ENTS RE8UIRED " +3 aqueous agar .ellosolve !(%ethoxyethanol# MET0OD " 2nfiltrate fixed tissues in +3 aqueous agar solution for " hour at +0%80:.. ( ;rientate tissues in an agar filled mould as indicated previously. 'llow the agar to set. Trim the block. * $ass the block through the following series, *0 minutes in each solution/ *03, +03, 903 ethanol) 703 ethanol plus cellosolve, (/") the same "/() pure cellosolve, * changes) cellosolve plus ester wax "/") pure ester wax, at least ( changes, the last overnight. 1 ,mbed as usual and cool rapidly. TEC0NICA- NOTE 6uzzell"1" dehydrates in dioxane, with amyl acetate as a transition solvent. 6locks are best cut using a retracting microtome. NITROCE--U-OSE 1 $ARA**IN 5A, DOU!-E IN*I-TRATION Aitrocellulose%paraffin wax double embedding is mainly used for brain, friable tissues and decalcified bone and is particularly useful for whole body sections of small animals and chitinous tissues. 2t combines the plasticity and support provided by nitrocellulose with convenient handling and microtomy of the paraffin technique. Tissues may be !a#infiltrated with thick nitrocellulose solutions and the resulting block trimmed, hardened in chloroform and infiltrated in paraffin wax, or !b#infiltrated with thin low viscosity nitrocellulose !?BA# solutions which are integrated into a normal processing schedule. $roprietary celloidin%ethanol%ether solutions provide a simple and convenient double%embedding method !Table "8#. The principle drawbacks of this technique are prolonged exposure of tissues to absolute ethanol and the high flammability and volatility of diethyl ether precluding machine processing. <se of methyl benzoate or methyl salicylate as ?BA solvents overcome these deficiencies. 2n $eterfi=s classic technique,"1( tissues are dehydrated to absolute ethanol, infiltrated with "3 celloidin in methyl benzoate with (%* changes over (1%9( hours !until clear#, hardened in three changes of toluene for 5 hours, then infiltrated as usual in paraffin wax.

olnar=s variation"1* !Table "9# is shorter and more logical, and is suitable for manual or machine processing. The n%butanol added to reduce the explosion hazard may contribute up to *03 of the weight of the nitrocellulose"(9 and must be taken into consideration when preparing solutions. Aitrocellulose dissolves slowly in methyl salicylate, and during preparation mixtures should be periodically shaken to assist dissolution. 2n recent times ?BA has largely replaced celloidin. Sections of double%embedded tissues may tend to wrinkle or curl on the waterbath. >loating on 7+3 ethanol or Cuyter=s fluid"*7 softens the ?BA and facilitates section flattening.

Met(ods for difficult tissues

0ARD DENSE TISSUES Tissues largely comprised of thickened keratin, dense collagen, closely packed smooth muscle fibres, colloid, areas of haemorrhage, thrombi or yolk, can be hardened excessively when processed on routine schedules and consequently, sections may crumble or shatter. 2deally the fixative, processing reagents, embedding medium and schedules should be selected to minimise hardening in these tissues. &espite careful processing hard tissues frequently require treatment with post%embedding ad@uvants before microtomy. ammalian tissues such as uterus, scirrhous carcinoma, leiomyomas and keratinised tissues are softened by fixing in 13 phenol in a mixture of absolute ethanol !9+ ml#, water !"0 ml# and chloroform !"0 ml#,(7 or by treating fixed tissues using 13 aqueous phenol for (1%9( hours.(7 Similar results are obtained by dehydrating tissues using phenol in the first bath of absolute ethanol, or in all dehydrant baths !Table "5#.*( Transition solvents such as chlorinated hydrocarbons and terpenes are recommended as they do not exacerbate tissue hardness. MUMMI*IED TISSUES ummified archaeological specimens for histology are first rehydrated then processed on a ?BA%paraffin wax double%infiltration schedule using phenol%ethanol to soften the tissues and amyl acetate as the transition solvent."11%"1+ Met(ods *or T(e Reco"er' Of Dried And Mummified Tissues REA ENTS RE8UIRED Solution !after Sandison"11# 'bsolute ethanol *0 ml >ormaldehyde, *93 0.+ ml Sodium carbonate 0.( g Water to "00 ml or Ban .leve J Coss= solution"1+

Trisodium phosphate 0.(+ g Water "00 ml MET0OD " 2mmerse tissues in either solution for (1%9( hours or more, depending upon the nature and thickness of the specimen !most tissues rehydrate and soften within 1%8 hours#. ( $rocess re%hydrated tissues on a normal schedule beginning in 903 ethanol. Tissues dried and hardened over years, and mummified archaeological specimens should be rehydrated in Sandison=s solution, then double infiltrated in phenol%amyl acetate%?BA% paraffin wax schedule. /O-K/ TISSUES Kolk%rich gonads, and muscle of marine and freshwater fish and invertebrates are routinely fixed in >''.. fixative !formaldehyde *93, "0 ml) glacial acetic acid, + ml) calcium chloride dihydrate, ".* g#"18 and processed by the schedule given in Table "7. This fixative has similar fixation image, processing and sectioning characteristics to 6ouin=s fluid, but without the hazard, cost and inconvenience of picric acid used in this fixative. 6uffered or saline formaldehyde fixatives cause excessive hardening of these tissues and are contra%indicated. Steedman"7 recommends polyester wax for minimising heat%induced hardening of difficult tissues. *ATT/ TISSUES >atty tissues such as breast or lipoma may be inadequately processed in what is normally a successful schedule for other tissues. ,thanol is a poor fat solvent. To ensure complete dehydration, a superior fat solvent such as acetone or isopropanol should be inserted before the final absolute ethanol, and chloroform or ",",",%trichloroethane used as the transition solvent. Ackno4ledgments The assistance of ?aurie Ceilly, Dary &oak and Savita >rancis in the preparation of this chapter is gratefully acknowledged. C,>,C,A.,S S'>,TK &'T'