Tissue processing

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.

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

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

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

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*+.to facilitate thin sectioning*0. $RESSURE AND . particularly lung. resulting in prolonged processing times. $rolonged immersion in paraffin wax at the correct temperature results in only slight tissue shrinkage"8. TEM$ERATURE 't low temperatures structural elements of tissues are stabilised against the destructive effects of solvent changes*+. dehydrant and transition solvent used"9. The extent to which tissues are affected during paraffin wax infiltration depends upon the combination of fixative. Biscosity and melting point of the infiltration medium partly determine the duration and temperature of processing conditions. Bacuum applied during dehydration. $ositive pressures for fluid transfer that are encountered in closed system processors are probably too low to have a significant influence on tissue infiltration. common an%isothermic techniques"7. Tissue shrinkage during infiltration in paraffin wax results mainly from the effect of heat on collagen"8. to 1+:. Tissues. The application of mild heat within the range *9:. pressure and agitation reduce the duration of tissue processing and improve the quality of infiltration. and the solvent boiling point is reduced. and concomitant reduction in the duration of tissue processing. .ACUUM -igh pressure facilitates infiltration of dense specimens with viscous resinous embedding media at the block forming stage"7. -eat increases the kinetic energy of molecules and rate of diffusion.*8. with stimulation of diffusion*1.((. clearing and infiltration stages improves the quality of processing. 2sothermally processed mammalian tissues show finer detail and less artefacts than those processed by the more practicable. muscle and yolk may harden and become brittle. thus facilitating evaporation of the reagent from the molten infiltration medium.*9 though tissues such as blood. Tissue%medium adhesion is enhanced if the embedding matrix has a fine uniform crystalline morphology which intimately contacts the tissue. with a corresponding decrease in solution viscosity. the key element of which is internal heating. <nfortunately at low temperatures reagent viscosities increase and diffusion rates decrease.*5 as well as the tissue type. $rocessing conditions Temperature.(" which can be avoided by maintaining embedding waxes (%*:. are de%aerated. but is rarely employed for biological specimens. &uration of wax infiltration is dependent upon viscosity and is not reduced by the application of vacuum*7. but may concomitantly increase shrinkage(". icrowave stimulated processing involves complex molecular interactions. above their melting points(". during the dehydration and clearing steps considerably reduces processing times"5..(*. -igh infiltration temperatures cause marked tissue shrinkage and hardening"9.

>or efficient and effective processing there should be a specimen volume to processing fluid volume ratio of at least "/+0. 2deally tissue cassettes should be placed in processors so that the cassette perforations are perpendicular to the fluid flow. . . 'lternate vacuum and positive pressure cycles during processing may provide some micro agitation within tissues. where the dehydrant. &ehydrants differ in their capacity to cause tissue shrinkage !>ig. acidified dimethoxypropane or diethoxypropane. remain static in the reagent. processing method. Ceagent diffusion time is therefore increased and if the duration of processing is not correspondingly increased. the most commonly used method. and economic factors. 2f tissues are allowed to settle on the bottom of a container. 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. &uring this procedure various cellular components are dissolved by dehydrating fluids. >or example.hoice of a dehydrant is determined by the nature of the task. 2n ultrasonic stimulated processing10%1" tissues and fluids are sub@ected to high frequency agitation and associated phenomena. Tissues are processed to the embedding medium by removing some or all of the free water. suspended and agitated within the medium to facilitate the exchange of dilute reagent from the tissues with the more concentrated reagent replacing it.hemical dehydration. Specimens are transferred through increasing concentrations of hydrophilic or water miscible fluids which dilute and eventually replace free water in the tissues. with simultaneous reduction in processing time. . or tidal action and flow of processing fluids ensures adequate fluid exchange. &ehydration is necessary in all infiltration methods. except where tissues are simply externally supported by an aqueous embedding medium. the embedding medium. inadequate processing will result. and water soluble proteins are dissolved in the lower aqueous alcohols1(. continual rotary or vertical motion of tissue containers. or are too tightly packed in the processor basket.A ITATION >luid interchange between processing reagents and tissues is promoted by exposure of the maximum tissue surface area to reagents. certain lipids are extracted by anhydrous alcohols. *#. 'gitation of tissues and fluids in manual processing is achieved using rotors or magnetic stirrers. 2n automatic tissue processors. &ehydration is effected as follows/ • • &ilution dehydration. Water is present in tissues in free and bound !molecular# forms. De('dration The first step in processing is dehydration. &uring processing. is hydrolysed by free water present in tissues to form acetone and methanol1*%+0 in an endothermic reaction. but this has yet to be substantiated. tissues should be loosely packed.

Tissues may be held and stored indefinitely in 903 ethanol without harm. De('drating agents A-CO0O-S These are clear. >ollowing fixation in anhydrous fixatives such as .+" incompletely fixed tissues may exhibit artefacts if placed directly in higher alcohols. "00 -igh Drade or Standard Drade# and as special ethylated Spirits !77.olloid. 2t is normally a poor lipid solvent except under microwave processing conditions*1. 6oth are satisfactory for histological purposes. blue when hydrated. when anhydrous. $rocessing times in absolute ethanol should be minimal. dehydration from aqueous fixatives is usually initiated in 803%903 ethanol.arnoy=s fluid. 2t is supplied as 77.thanol dissolves nitrocellulose slowly unless combined in equal proportions !or better. .((%(*. The salt is self%indicating/ white when anhydrous.thanol is a rapid. following any necessary post fixation treatment. 2t is prepared by carefully heating . then two or three changes of absolute ethanol before proceeding to the clearing stage. delicate specimens are dehydrated in a graded ethanol series from water through "03%(03%+03%7+3%"003 ethanol.2n the paraffin wax method. efficient and widely applicable dehydrant. "/(#+* with diethyl ether.thyl alcohol formulations differ in standards and nomenclature worldwide and it may be necessary to consult various tables to ascertain the ethanol concentration. $rogressive removal of bound water from carbohydrates and proteins during prolonged immersion in absolute ethanol causes tissues to harden excessively and become brittle"7. miscible with water and.*8 &uration of dehydration should be kept to the minimum consistent with the tissues being processed. Et(anol is probably the most commonly used dehydrant in histology. collagen and yolky tissues are particularly affected"7.5+3 ethanol !absolute ethanol. . are used in a similar manner to that described for ethanol. Whilst well fixed tissues can be transferred directly to 7+3 ethanol. The problem is exacerbated by heat during wax infiltration. progressing through 703%7+3 ethanol. for example dehydration is initiated in "003 ethanol. . . colourless. and is only slightly soluble in ethanol. Tissue blocks " mm thick should receive up to *0 minutes in each alcohol. including universal solvents.5+3 ethanol denatured with (3 methanol#. though generally in different concentration increments. The dehydrant concentration at which processing is initiated depends largely upon the fixative employed. alcohols also act as secondary coagulant fixatives during tissue processing. . 'nhydrous cupric sulphate added to the final absolute ethanol on a tissue processor scavenges any water present. blocks + mm thick require up to 70 minutes or longer in each change. hydrophilic liquids. flammable. 2n addition to their role as dehydrants. To minimise tissue distortion from diffusion currents. with most organic solvents.ther dehydrants. blood.

fixed tissues are transferred via 803%903 isopropanol or ethanol to absolute isopropanol. isopropanol is used as a transition solvent following ethanol dehydration*1. 2t causes less hardening and shrinkage than ethanol.(* is a less costly substitute for n% butanol. these reagents do not act as secondary fixatives. flammability and cost. is fully miscible with melted paraffin wax++. dense tissues. 2t is flammable. 'nhydrous calcium sulphate !&rierite# or molecular sieves act in a similar manner but are non%indicating. on a hotplate at (+0:.8" -/CO-1ET0ERS <nlike the alcohols. 2n microwave processing it tends to harden tissues more than ethanol*1.53 !absolute# isopropanol. Met(anol is a good ethanol substitute+1 but rarely used for routine processing because of its volatility. Normal and tertiar' %utanols are universal solvents mainly used for small%scale manual processing of plant and animal tissues in teaching and research. and the vapours are eye irritants. with a penetrating camphor%like odour. 2t is a poor lipid solvent.80 though this is offset by the prolonged processing schedules which may result in tissue shrinkage.hydrated cupric sulphate until it turns white. which can remain in the solvent for extended periods without harm. slightly slower in action and not as hygroscopic as ethanol. Isopropanol was first suggested as an ethanol substitute during the prohibition era in the <nited States+1.. 2n microwave stimulated processing. and is readily expelled from tissues and wax baths. Tertiary%butanol is widely used in plant histology8" but rarely for animal tissues"9. a ma@or disadvantage. To minimise shrinkage. . though unsatisfactory as a dehydrant. 2sopropanol shrinks and hardens tissues less than ethanol+1%+9 and is used to dehydrate hard."5.ool in a desiccator. Solid drying agents are placed in a "%( cm thick layer in the reagent container. with similar properties and processing characteristics((. and will not dissolve nitrocellulose unless mixed with acetone. and apart from solvent effects do not appear to alter tissue reactivity. .(( A%butanol is poorly miscible with water and only slowly miscible with paraffin wax. and covered with filter paper or fine sponge to avoid mixing with the specimens during tissue agitation.*5. 2sopropanol only dissolves nitrocellulose in the presence of esters+7 such as methyl benzoate or methyl salicylate. but is not used in staining work stations as many other dyes are insoluble in this solvent. 2so%butanol. 2sopropyl alcohol has also been recommended as a xylene substitute+5. 2t is a universal solvent available as 77. 2n processing it is used in a similar manner to n%butanol. 6elow (8:. and is used in methyl salicylate%based double%infiltration methods. 2sopropanol is a solvent for some lipid%soluble dyes.(*. Aormal butanol is recommended for processing lightly chitinised arthropods*5 and rodent tissues. it is hygroscopic crystalline solid. 2t cannot be used as a dehydrant in alcohol%ether%celloidin techniques. 2sopropanol is completely miscible with water and most organic solvents. but a far superior lipid solvent.

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

"5 The term clearing arises because some solvents have high refractive indices !approaching that of dehydrated fixed tissue protein# and.+" 'cidified & $4&. 'cetone is not recommended for microwave processing as it causes excessive nuclear shrinkage*1. . The reagent is stored at 1:. alcohol and most organic solvents except petroleum ethers.n exposure to air and light. and is comparable to conventional dehydration for tissue morphology and staining reactions.1*%+" They are flammable and form peroxides. or clearant# miscible with both the dehydrant and the embedding medium is used to facilitate the transition between dehydration and infiltration steps.*8 The coupling action permits tissues and celloidin sections to be cleared from lower strength alcohols.90 This property is used to ascertain the endpoint and . phenol and its solutions develop a pink to reddish discolouration.+" . The solvent dehydrates rapidly causing little shrinkage or hardening. & $ shrinks tissues slightly less than &. 2n the final stage shrinkage may result from the extraction of fat by the transition solvent.15 a transition solvent such as methyl salicylate17 or toluene should precede infiltration with wax.reosote and aniline are used less commonly though in a similar manner to phenol. -owever a transition solvent is normally interposed before the paraffin baths. and from transition solvent to wax"9. as in Weigert=s carbol%xylol !xylene 9+ ml and phenol (+ ml#. Tissues are processed as in dioxane method. . and is possibly the best of the universal solvents. 2t is soluble in water.ontainers must be protected from light and tightly sealed. most organic solvents. but not most dyes. $henol consists of clear hygroscopic acicular crystals and is also available as 503 w4w liquefied phenol. . $(enol. on immersion. any dehydrants are immiscible with paraffin wax.oncentrated solutions coagulate nitrocellulose.hemical dehydration is suitable for rapid manual processing or machine processing. Clearing .$. is not wax miscible and a post dehydration rinse in acetone. 2&2 dimet(o)'propane !& $# and (. Tetrahydrofuran can form explosive peroxides which renders solvent recovery distillation dangerous88.$ can be reused several times.( diethoxypropane !&. though dehydration times need to be extended. 2t is completely miscible with water."5 !>ig 1#. beechwood creosote and aniline facilitate dehydration when mixed with transition solvents.$ are miscible with paraffin wax however methanol.Tissues can be transferred directly from acetone to paraffin wax as the solvent boils off under vacuum85. one of the hydrolysis products. anhydrous tissues are rendered transparent or clear. $henol crystals and 503 concentrate react violently with formaldehyde.$# are used for chemical dehydration of tissues. Shrinkage occurs when tissues are transferred from the dehydrant to the transition solvent. 2t dissolves mountants. highly volatile and flammable universal solvent with an offensive ethereal odour.87 2t is less toxic than dioxane for which it can be substituted. in a spark%proofed refrigerator. paraffin wax and mounting media. .learing is the transition step between dehydration and infiltration with the embedding medium.+0 & $ and &. ante medium. Tetra('drofuran is a colourless. and a solvent !transition solvent.

miscible with most organic solvents and with paraffin wax. odorous mercaptans and hydrogen sulphide. and do not render tissues transparent. Toxic effects of petroleum solvents are broadly similar to those of pure hydrocarbons % skin degreasing. do not render tissues transparent and the clearing endpoint !generally when the specimen sinks in the solvent# must then be determined empirically. . 6lends with high aromatic and naphthene but reduced paraffin content such as Shell E*69". Tissues stabilised in protein coagulant fixatives !6ouin=s or S<S'# are less affected. -owever. high flash%point solvents. They coagulate nitrocellulose. 2ndustrial grade xylene may contain nearly (+3 of other solvents such as ethyl benzene. moderately toxic.(* 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.88 any of these solvents have a particularly strong petroleum odour which some people find ob@ectionable. Transition sol"ents 00/DROCAR!ONS These are odourless flammable liquids with characteristic petroleum or aromatic odours. Toluene and )'lene clear rapidly and tissues are rendered transparent. facilitating clearing endpoint determination. acute intoxication and narcosis in high concentrations. are good. Those with high paraffin but little or no naphthene and aromatic content often have low flammability and toxicity. . clear more slowly than xylene and toluene. but otherwise do not alter tissue reactivity nor behave as secondary fixatives during processing. $etroleum sol"ents have a gentle. 6lends of aromatic. The presence of opaque areas indicates incomplete dehydration. non%hardening action on tissues. flammability and solvent action# can be used as xylene substitutes. ."9. vacuum and pressure safety factors cost and convenience. naphthenic and aliphatic solvents !each with varying toxicity. and a slow and gentle clearing . Eylene hardens tissues fixed in non%protein coagulant fixatives and prolonged clearing in the solvent should be avoided.nly the sulphur and benzene%free solvent%grade xylene should be used for histological purposes.hoice of a clearing agent depends upon the following/ • • • • • the type of tissues to be processed. notably chlorinated hydrocarbons. other solvents. Transition solvents extract certain tissue substances such as lipids. and the type of processing to be undertaken the processor system to be used intended processing conditions such as temperature.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. with traces of benzene.duration of the clearing step.

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

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

Tissues may remain in it indefinitely without harm. Em%edding is the process by which tissues are surrounded by a medium such as agar. 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. Barious substances have been used to infiltrate and embed tissues for microtomy. and 80:. elasticity. tissues can be infiltrated with a solution of a substance dissolved in a solvent. . plasticity. which solidifies on evaporation of the solvent to provide a firm mass suitable for sectioning. but not always. viscosity and adhesion and should be harmless to the embedded material. 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 media are selected according to the nature of the task for which they are required. Some of the specific safety requirements relating to processing solvents are summarised in Table *. 2t clears tissues from 503%703 alcohol with minimal hardening. the medium in which they are finally embedded. 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. which is fluid when hot and solid when cold. ?ike the other terpenes it is difficult to eliminate from paraffin wax. or wax which when solidified will provide sufficient external support during sectioning. Tissues are infiltrated by immersion in a substance such as a wax. Infiltration is the saturation of tissue cavities and cells by a supporting substance which is generally. Aone completely fulfil the foregoing criteria. for example nitrocellulose in alcohol%ether. gelatine. 'lternatively.Terpineol is a clear almost colourless mixture of isomers with a faint pleasant odour and very low evaporation rate.

+3 ceresin. Small. . -igh molecular weight mixtures melt at higher temperatures than waxes comprised of lower molecular weight fractions. modified to provide improved tissue support and sectioning qualities."%+3 beeswax. 2t is about two thirds the density and slightly more elastic than dried protein. asphalt.** Wax hardness !viscosity# depends upon the molecular weight of the components and the ambient temperature. rubber. There is consequently less deformation during thin sectioning.59 .51%58 MODI*IED $ARA**IN 5A. then infiltrated a second time with wax in which they are also embedded. $araffin wax is traditionally marketed by its melting points which range from *7:. odern tissue processors are equipped to deliver vacuum. Tissue%wax adhesion depends upon crystal morphology of the embedding medium. consisting of wax baths. or phenanthrene. 0.. reduces the time tissues are sub@ected to high temperatures thus minimising heat%induced tissue hardening. and prolongs the life of wax by reducing solvent contamination. to 85:. more homogeneous wax with good cutting characteristics. facilitates complete removal of transition solvents.rystalline morphology of paraffin wax can be altered by incorporating additives which result in a less brittle. The procedure assists the complete and rapid impregnation of tissues with wax. bayberry wax. Setting temperature does not appreciably affect crystal size.acuum infiltration is the impregnation of tissues by a molten medium under reduced pressure. $arffin 4a) $RO$ERTIES $araffin wax is a polycrystalline mixture of solid hydrocarbons produced during the refining of coal and mineral oils. or vacuum and pressure. In"estment generally refers to the practice of embedding wax infiltrated tissues in another wax.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. uniform sized crystals provide better physical support for specimens through close packing. . to which a vacuum of up to 980 mm -g is applied using a water or mechanical pump. such as $iccolyte%paraffin wax. fluid trap and vacuum gauge. to all or some reagent stations during the processing cycle. Bacuum infiltration requires a vacuum infiltrator or embedding oven.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.

a supply of moulds in which to embed the tissues. 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 " . check against worksheet entry to ensure the correct number of tissue pieces are present. Dimet('l sulp(o)ide !& S. embedding oven and ice will suffice. there should be sufficient room for the tissue with allowance for at least a ( mm surrounding margin of wax.# added to proprietary blends of plastic polymer paraffin waxes reduces infiltration times and facilitates thin sectioning. '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.mbedding requirements and procedures are essentially the same for all waxes.55%57 have largely been replaced by uniform. above its melting point.pen the tissue cassette. . filtered paraffin wax held at (%1:. Some individuals who handle & S. metabolites.therwise a wax dispenser. a cold plate to rapidly cool the wax..% paraffin wax may experience an unpleasant and annoying oyster or garlic taste probably caused by & S.% paraffin wax88 are minimal if correct laboratory hygiene is practised. 't the completion of processing. . These elements are conveniently combined in commercially available embedding stations !>ig +#.7+ $ossible health risks associated with the use of & S. Cequirements for embedding are as follows/ • • • a supply of clean. 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. $lastic pol'mers such as polyethylene wax. added to improve adhesion. tissues are held in clean paraffin wax which is free of solvent and particulate matter. and only the technique for paraffin wax is provided here in detail.arly histological wax formulations*8. scavenges residual transition solvent and probably alters tissue permeability by substituting for or removing bound water thus improving infiltration.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* $olymer waxes are incorporated in the ma@ority of proprietary histological paraffin wax blends presently available.71 & S. high quality proprietary blends of histological paraffin waxes. ( Select the mould.7"%7( $iccolyte mixtures cannot be used in certain models of fluid%transfer type tissue processors. hardness and plasticity.

>or routine purposes tissues are most conveniently processed through dehydration. Some general considerations !>ig. 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. + . This ensures that the correct orientation is maintained and the tissue surface to be sectioned is kept flat. and not placed at random. <sually tissues are embedded with the surface to be cut facing down in the mould. There are two broad types of automatic tissue processors % tissue%transfer and fluid%transfer types."9 This compression is almost fully recovered when sections are floated on a warm waterbath8) compression resulting from microtomy is only partially recovered. paraffin wax shrinks up to "+3. $rocessing met(ods and routine sc(edules Tissues are usually more rapidly processed by machine than by manual methods. 5 Cemove the block from the mould. Automated tissue processing TISSUE1TRANS*ER $ROCESSORS These processors are characterised by the transfer of tissues. and was . 7 .9# are as follows/ • • • • elongate tissues are placed diagonally across the block tubular and walled specimens such as vas deferens. clearing and infiltration stages automatically by machine.* >ill the mould with paraffin wax. through a series of stationary reagents arranged in%line or in a circular carousel plan.ross check block. 2ncorrect placement of tissues may result in diagnostically important tissue elements being missed or damaged during microtomy. 8 2nsert the identifying label or place the labelled embedding ring or cassette base onto the mould. 1 <sing warm forceps select the tissue. causing compression in tissues. The rotary or carousel is the most common model of automatic tissue processor. 2n circumstances where precise orientation is essential tissue should be marked or agar double embedded.ool the block on the cold plate. or carefully submerge it under water when a thin skin has formed over the wax surface. orienting the tissue and firming it into the wax with warmed forceps. label and worksheet. contained within a basket.hill the mould on the cold plate. ORIENTATION O* TISSUE IN T0E !-OCK . although the latter can be accelerated by using microwave or ultrasonic stimulation. 9 . taking care that it does not cool in the air) at the same time. &uring cooling. cysts and gastrointestinal tissues are embedded so as to provide transverse sections showing all tissue layers tissues with an epithelial surface such as skin.orrect orientation of tissue in a mould is the most important step in embedding.

*-UID1TRANS*ER $ROCESSORS 2n fluid%transfer units !>ig. >luid%transfer processors overcome the main drawbacks of the tissue%transfer machines. pin or touch pad programmed. and vacuum%pressure options for each station. metal%corrosive fixatives. Cepresentative schedules for rapid and overnight processing are provided in Table +.7# processing fluids are pumped to and from a retort in which the tissues remain stationary. for example chloroform.. or wax additives such as $iccolyte. *%1 paraffin wax stations with variable temperature settings between 15%85:. with a capacity of *0%""0 cassettes depending upon the model. $rocessors are provided with alert systems and diagnostic programs for troubleshooting and maintenance. Tissues should not be packed too tightly in baskets so as to impede fluid exchange.97 2t is provided with 7%"0 reagent and (%* wax positions. ENERA. a wide range of solvents. There are "0%"( reagent stations with temperatures ad@ustable between *0%1+:. Schedules are microprocessor programmed and controlled. Tissues accidentally returned into fixative or alcohol following wax infiltration are recovered by methods outlined in Table 8. 2n more recent models !>ig.CONSIDERATIONS 6askets and metal cassettes should be clean and wax%free. $rocessing schedules !Table 1# are card%notched. $rocessors must be free of spilt fluids and wax accumulations to reduce hazards and to ensure mechanical reliability. processing reagent changes. Timing and delay mechanism must be correctly set and checked against the appropriate processing schedule.invented by 'rendt in "707.. >luid levels must be higher than the specimen containers. certain solvents. temperature checks on the wax baths and the completion of the routine . 'gitation is achieved by tidal action. These machines have a rapid turn%around time for day4night processing. TISSUE RECO. Some models are unable to accept mercury or dichromate%based fixatives. and relatively viscous nitrocellulose solutions can all be accommodated. in particular. Bacuum%pressure cycles coupled with heated reagents allow effective reductions in processing times and improved infiltration of dense tissues. Tissue%transfer processors allow maximum flexibility in the choice of reagents and schedules that can be run on them. Tissues are unable to dry out within the sealed retort and reagent vapours are vented through filters or retained in a closed%loop system. &epending upon the model these machines can process "00%*00 cassettes at any one time. ' processor log should be kept in which the number of specimens processed. >luid agitation is achieved by vertical oscillation or rotary motion of the tissue basket.5# the tissue basket is enclosed within an integrated fume hood during agitation and transfer cycles thus overcoming the disadvantages of earlier styles.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.

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

"0# which is fitted with precise temperature control and timer. .solvent. 0INTS *OR MICRO5A. ' turntable or in%built radiation disperser facilitates even reagent heating. 'n alternative approach is to carefully layer the dehydrant onto the transition solvent and introduce the tissue into the upper layer.ATION >or rapid processing. . Ceagent volumes should be at least +0 times that of specimen volume.D 100 !1+ ml#"0( from which specimens can be transferred directly to dehydrant. and an interlocked fume extraction system to preclude accidental solvent vapour ignition. and net power levels at various settings must be determined before the oven is used to process tissues. &omestic microwave ovens with a temperature probe and timer accurate to seconds are suitable for tissue processing.*1 • • $rocess blocks of similar thickness together. .*1 E8UI$MENT Tissues are processed in conventional plastic cassettes. 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.79%"0" $rocessing is undertaken in a dedicated microwave oven !>ig.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. $rocessing times for formaldehyde%fixed tissues need to be increased above those provided for coagulant%fixed tissues. Ceagents are carefully decanted and the specimen placed in a fresh change of transition solvent. 'gitation is provided by an air%nitrogen system. duration of cycle time.*1 $rocessing schedules are provided in Table "(.7 or in 7+3 ethanol !800 ml#%polyethylene glycol $. 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.alibration of domestic ovens is essential for optimum results and the accuracy of the temperature probe. tissues are fixed by microwave irradiation.vens should therefore be used within a fume cupboard to minimise this problem.E $ROCESSIN % Tissue blocks should be as thin as possible. The tissue sinks as the dehydrant gradually replaces the transition solvent.*1 $icric acid fixed tissues should not be microwave processed as there is an explosion risk even in well washed tissues. >ormaldehyde%fixed tissues must be rinsed in running tap water for + minutes before microwave processing and an extra dehydration change incorporated in the schedule. ?ength and width are not as important.*1. tissues will remain submerged throughout the clearing stage. including those with !provided the metal lids lie below the fluid#. *I.

or preferably methyl benzoate or methyl salicylate !Table "*#. <nfixed tissue blocks "%( mm thick can be fixed and processed to paraffin wax using ultrasonic%stimulation in " hour 1+ minutes. $rocessing is performed in reagent containers suspended in a detergent solution within the transducer tank of an ultrasonic cleaner operated at +0 watts. A8UEOUS MEDIA Agar has a high melting point and low gelling temperature of agar make it ideal for . 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.ells and organelles such as cilia. Cesin embedding methods are now used for many of these applications. 'n immersion heater is used to elevate bath temperatures for paraffin wax infiltration. tissue processing for electron microscopy.• • • • The temperature probe should be placed centrally in processing baths. '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. $re%heat paraffin wax baths in a conventional oven.10 Tissues are dehydrated in ethanol and cleared in toluene. <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. Ultrasound1stimulated processing <ltrasonics are used in histopathology to accelerate fixation. improving the sensitivity of immunohistochemical reactions. 't lower frequencies cavitation phenomena and attendant heat. conventional staining and for accelerated tissue processing. .oagulant fixatives provide optimal stabilisation for ultrasonic%stimulated processing. 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. Aon%resinous embedding media include those listed below. pressure and streaming effects may damage tissues and care must be exercised. . tissue softening in post%embedding ad@uvants. . Tissues are placed in metal or plastic cassettes for processing. the decalcification of bone.ld and friable specimens sometimes exhibit marginal distortion and erosion. microvilli and desmosomes are all well preserved.10%1" The most important effect of ultrasound at frequencies of "00 k-z%" -z is agitation.

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

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

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

( 'dd formaldehyde and mix well. amyl acetate. 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. mixtures of di% and tri% nitrocellulose. 2mmunohistochemical investigations such as immunophenotyping of lymphoid and non%lymphoid cells"(5 are possible on nitrocellulose processed tissues. 2t is highly flammable. Aitrocellulose is insoluble in water."(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. A AR1$ARA**IN 5A. are used when tissues require external support or particular pre%embedment orientation.0 g ( &istilled water 70 ml * *93 formaldehyde solution "0 ml MET0OD " &issolve the agar in the distilled water using a boiling water bath. in methyl benzoate as it is explosive if detonated when dry.. $araffin wax double infiltration methods provide hard tissues with additional support provided by substances such as agar or nitrocellulose. . and must be kept alcohol%damped with n%butanol or as 53 solutions in ethanol%ether or "3 ?BA.# and ?ow Biscosity Aitrocellulose !?BA#. 0/DRO$0O!IC MEDIA Nitrocellulose . These methods and principles present possibilities for immunohistochemistry. Store at 1:."(1 has superior penetration and final block hardness and is supplied in various grades of viscosity and nitrogen content. but soluble in absolute ethanol%diethyl ether.elloidin !. Tissues are processed at room temperature producing minimal and shrinkage and hardening. Dou%le em%edding and dou%le infiltration met(ods &ouble embedding methods such as agar%paraffin embedding. but appear to have received little attention. are composed of yellowish%white matted filaments with the appearance of raw cotton. methyl salicylate and (%ethoxyethanol and is set by most hydrocarbon solvents. * &istribute (0 ml aliquot=s into screw capped bottles. with the convenience and ease of wax microtomy. ."*8 Agar Em%edding Medium REA ENTS RE8UIRED " 'gar !technical or microbiological grade# ".formal%polyester wax and mercury%formal%polyester wax mixtures. which can be lost during tissue processing.89 Aitrocellulose tissue processing techniques are generally employed for sectioning hard tissues such as bone. autoclave or microwave oven."(9 or for delicate embryonic material. ?BA tolerates up to 83 of water.+*.elloidin solutions have a low tolerance of water and dehydration must be thorough.+ % *. methyl benzoate."(+%"(8 for topographical studies of central nervous system tissues.

* $lace specimen in agar%filled mould and orientate.mbed tissues by pipetting agar solution over tissue fragments correctly oriented on a clean flat surface"(7 or membrane filter. rinse with water. 8 $ipette "%( ml of agar. . Trim and notch the agar block as required. ?oosen container caps before remelting agar. 7 <sing fine forceps.ollodion !add 13 nitrocellulose to (+ ml absolute ethanol / 9+ ml diethyl ether# MET0OD " $lace "%( ml of collodion into a "0 ml glass centrifuge tube. 5 'llow the mass to cool for + minutes. moulds made from cut%down plastic syringes. Cotate the tube so that an even layer of collodion coats and sets on the inside surface) gentle blowing into the tube hastens the setting process. ( >ill the tube with 903 ethanol and stand for + minutes to harden the collodion. detach the specimen from the mould by sliding a scalpel down one side. Agar1$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:. oven. or by immersing blocks in 903 ethanol or fixative for "%* hours."*0 or in metal Tissue%Tek type moulds. &rain the ethanol.1 >or use melt the agar as previously indicated. + ."*(.."0+ TEC0NICA. $rocess normally."*+ ( <nstabilized agar embedments sometimes disintegrate during processing and must be stabilised by the addition of (. * 'dd fixed aspirate or suspension to tube.. 1 .+3%13 formaldehyde to the medium. cross checked with request slip and specimen container.NOTES " Specimens can be embedded in silicon%rubber flat embedment moulds. 1 'llow agar to solidify for + minutes. carefully detach the collodion bag and contents from the tube. cooled to approximately 1+:. + &ecant supernatant fluid. $lace embedding cassette on top of the mould to identify the tissue. Agar1$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 . into the centrifuge tube. and hold in a 80:. leaving a *%+ mm width of agar surrounding the tissue.entrifuge at (000 rpm for *0 seconds. + When the agar block is solid. cool to +0%80:. 9 Capidly resuspend the specimen and centrifuge at (000 rpm for " minute.. ( Aumber embedding cassette.

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

colloid. 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. dense collagen. 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. and during preparation mixtures should be periodically shaken to assist dissolution. water !"0 ml# and chloroform !"0 ml#.(7 Similar results are obtained by dehydrating tissues using phenol in the first bath of absolute ethanol.leve J Coss= solution"1+ . closely packed smooth muscle fibres. scirrhous carcinoma. and is suitable for manual or machine processing. 2deally the fixative. or in all dehydrant baths !Table "5#. 2n recent times ?BA has largely replaced celloidin. can be hardened excessively when processed on routine schedules and consequently.*( Transition solvents such as chlorinated hydrocarbons and terpenes are recommended as they do not exacerbate tissue hardness. areas of haemorrhage. Aitrocellulose dissolves slowly in methyl salicylate. ammalian tissues such as uterus."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. embedding medium and schedules should be selected to minimise hardening in these tissues.+ ml Sodium carbonate 0.olnar=s variation"1* !Table "9# is shorter and more logical. &espite careful processing hard tissues frequently require treatment with post%embedding ad@uvants before microtomy. >loating on 7+3 ethanol or Cuyter=s fluid"*7 softens the ?BA and facilitates section flattening. leiomyomas and keratinised tissues are softened by fixing in 13 phenol in a mixture of absolute ethanol !9+ ml#.( g Water to "00 ml or Ban . *93 0.(7 or by treating fixed tissues using 13 aqueous phenol for (1%9( hours. sections may crumble or shatter. Met(ods for difficult tissues 0ARD DENSE TISSUES Tissues largely comprised of thickened keratin. processing reagents. thrombi or yolk. Sections of double%embedded tissues may tend to wrinkle or curl on the waterbath.

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

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