tissue preparation for immunocytochemistry · keywords: immunocytochemistry; fixation; tissue...

42 Clin Pathol 1997;50:422-428

Tissue preparation for immunocytochemistry

J H Williams, B L Mepham, D H Wright

AbstractAims-To investigate the effect of tissuepreparation on immunostaining and toestablish whether there is a standardtissue preparation schedule that allowsoptimal demonstration of all antigens.Methods-Blocks of tonsil were subjectedto variations to a standard fixation,processing, and section preparationschedule. The sections were stained withfive antibodies-L26 (CD20), UCHL1(CD45RO), CD3, vimentin, and anti-kappa light chain-using the streptavidin-biotin immunostaining technique. Whenfurther investigation was necessary, othertissues and antibodies were used andwhere weak immunostaining was obtainedthe use of microwave pretreatment toimprove staining was tested.Results-Several factors involved in fixa-tion were found to affect immunoreactiv-ity. These included the duration, pH, andtype of fixative used. In tissue processingonly temperature and the duration of thedehydration and wax infiltration stepsaffected immunoreactivity. Of all thefactors investigated, the temperature andduration of the section drying had thegreatest effect. In contrast, long termstorage of cut sections before immuno-staining had no effect on the reactivity ofthe antibodies tested. Antibodies werefound to be affected by alterations totissue preparation by varying degrees,UCHL1 and vimentin being the most sus-ceptible to changes in fixation and L26 tochanges in processing. Where weak stain-ing occurred, microwave pretreatmentwas generally found to eliminate-the prob-lem.Conclusions-There is no standard tissuepreparation schedule for the optimaldemonstration of all antigens. Factorsinvolved in all aspects of tissue prepara-tion can affect immunoreactivity, so it isimportant that precise details of thepreparation schedule are given when re-porting immunocytochemical studies,rather than using the general term "rou-tinely fixed and processed".( Clin Pathol 1997;50:422-428)

Keywords: immunocytochemistry; fixation; tissuepreparation

The most common cause of poor immuno-staining in paraffin wax embedded tissue is notthe technique itself but the alteration of theantigenic sites by the initial process of tissuepreparation' which incorporates fixation,

processing, and the preparation of the tissuesections. The establishment of a tissue prepara-tion schedule for optimal immunostaining ofall antigens has been a problem since immuno-cytochemical staining of paraffin embeddedtissue was first developed.2 Since that time sev-eral studies have been undertaken to investi-gate this problem, although these have mainlybeen concerned with fixation rather than withtissue processing or section preparation.

Tissue fixation has a significant influence onimmunostaining as most antigens are alteredduring the process. Fixation involves a numberof variables including the type, concentration,pH, and osmolarity of the fixative, and theduration and temperature of the fixation proc-ess; all ofwhich can affect immunocytochemis-try. Other factors reported to affect fixationand therefore possibly immunoreactivity arethe volume of fixative,3 delayed fixation,4 orsecondary fixation by alcohol-either duringfixation by methanol which is added as apreservative to commercially preparedformalin9 or in alcohol during processing iffixation is incomplete.'0-13

In contrast to the number of studies on theeffect of fixation on immunoreactivity, investi-gations into the effect of tissue processing havebeen limited; weak immunostaining because ofpoor tissue preparation being blamed on inad-equate fixation rather than on other aspects oftissue preparation. It was reported in the late1980s, shortly after the development of theenclosed tissue processor, that the use of thissystem could cause problems with both tissueand antigen preservation. This was eitherthrough carryover of reagents from one step tothe next, in particular xylene from the waxcleansing cycle at the end of the programmeinto the first step of the cycle,14-16 or throughthe use of inferior quality xylene duringprocessing.'5 The type of reagent and thetiming and temperature used for each step ofprocessing (dehydration, clearing, and waxinfiltration) have also been reported to affectimmunoreactivity.'7-22 Other factors which canaffect processing and therefore possibly immu-noreactivity are the use ofxylene as a substitutefor water to make up graded alcohols,23 and theapplication of vacuum during processing.24No studies have exclusively investigated the

effect on immunoreactivity of section prepara-tion, which includes wax embedding, sectioncutting and drying, and subsequent sectionstorage. It has been suggested that the use oflower drying temperatures or shorter times25-29and the avoidance of long term storage ofcut sections can help preserve immuno-reactivity.3032

UniversityDepartment ofPathology,Southampton GeneralHospital,Southampton S0166YD, United KingdomJ H WilliamsB L MephamD H Wright

Correspondence to:Mrs J H Williams.

Accepted for publication26 February 1997

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The introduction of antigen retrieval byheating tissue sections in a microwave" or

pressure cooker34 3 before immunostaining hasbeen a major breakthrough in improving weakimmunoreactivity, particularly in suboptimallyprepared tissue."2 34

The aim of this study was to investigate allaspects of tissue fixation, processing, andsection preparation and their effect on subse-quent immunostaining, to establish a standardtissue preparation schedule which would allowoptimal staining of all antigens. This was

carried out using primary antibodies whichfrom experience were known to be fixation or

processing dependent to varying degrees.

MethodsTISSUE PREPARATIONTonsils were obtained fresh from the operatingtheatre, cut into 2 mm slices, and subjected toa number of variations to a standard fixation,processing, and section preparation protocol.Each block was subjected to variation of onlyone of the three components of tissue prepara-

tion, the other two remaining standard. Thestandard tissue preparation protocol com-

prised fixation for 24 hours in 10% neutralbuffered formalin (NBF) at room temperaturein 50 ml of fixative, followed by processing on a

16 hour overnight schedule at ambient tem-perature on a Shandon Hypercenter 2 (LifeSciences International, Basingstoke, UK)(table 1). Paraffin sections were cut at 4 mm,mounted on 3-aminopropyltriethoxysilanecoated slides, and dried at 37°C overnight.

Variations to fixation protocol(1) Different types of fixative:

(a) 10% NBF36;(b) 10% formalin in tap water36;(c) 10% formal saline36;(d) 10% neutral buffered formalin with

saline (NBFS);(e) 10% formal acetic37;(f) 10% zinc formalin38;(g) Carson's fixative39;(h) Bouin's fixative36;(i) B5.4°

(2) Period of fixation: < 5, 5, 12, 24, 36, and48 hours.

(3) Temperature of fixation: 18°C, 270C, and370C.

(4) pH of 10% formalin: pH 3.0, 5.0, 7.0, 9.0,and 11.0.

(5) Delay before fixation: 0, 1, 2, 4, and 8hours.

(6) Different volumes of formalin: ratio of tis-sue:formalin of 1:1 to 1:20.

(7) Addition of methanol to formalin: 0, 0.25,0.5, 1, 1.5, 2, and 5%.

Variations to processing protocol(1) Comparison between the carousel type

and the enclosed vacuum processor.(2) Comparison of different clearing agents

with xylene:(a) chloroform;(b) Clearene (Surgipath, St Neots, UK);(c) xylene substitute (Shandon).

(3) Comparison of different paraffin waxes:

(a) non-polymer (Ames Tissue Tek II,Bayer, Newbury, UK);

(b) polymer (Ames Tissue Tek III, Bayer);(c) polymer + DMSO ("Paramat extra",

Merck, Poole, UK);(d) microcrystalline (Fibrowax, Merck).

(4) Reduced and extended processing timesfor dehydration, clearing and wax infiltra-tion.

(5) Temperature of processing: ambient and450C.

(6) Use ofvacuum during all processing steps.(7) Number of blocks and position of tissue in

(8)(9)

the carrier during processing.Xylene contamination of formalin.Comparison between repeated use of highand lower quality xylene for clearing.

Variations to section preparation protocol(1) Dried at either 37°C overnight in a section

drying oven, 60°C for 1, 4, or 8 hours andovernight in an incubator, or face down ona section drying hotplate at approximately70°C for 0.5, 2, and 8 hours.

(2) Dried at 37°C overnight and stored for 1day, 1 week, 1, 3, and 6 months at roomtemperature and 4°C before immunostain-

Table 1 Standard processing schedule

Station Reagent Temperature Vacuum Time (h)

2 70% alcohol A Y 13 90% alcohol A Y 14 100% alcohol A Y 0.55 100% alcohol A Y 16 100% alcohol A Y 27 100% alcohol A Y 28 Xylene:alcohol

(50:50) A Y 19 Xylene A Y 1.510 Xylene A Y 1.511 Lambwax

(RA Lamb,London, UK) 60°C Y 1.5

12 Lambwax 600C Y 3Total processing

time 16

A, ambient temperature.

Table 2 Main panel ofprimary antibodies used

Pre-Antibody CD No Supplier Cat No Dilution treatment

L26 CD20 Dako M755 1/1000 NTUCHL1 CD45RO Dako M742 1/100 NTCD3 CD3 Dako A452 1/200 +TVimentin Dako M7020 1/1000 +T

(3B4)Kappa Dako A191 1/500 +T

NT, no pretreatment; +T, trypsin pretreatment.

Table 3 Additional antibodies usedfor section preparationexperiments

Antibody Supplier Cat No Dilution Pretreatment

MIBI BindingSite* 0505 1/1000 +MW

PC1O Giftt - 1/800 +T1D5 Dako M7047 1/500 +MWBCL-2 Dako M887 1/150 +MWaSMA Dako M851 1/2000 +MWCEA Dako M7072 1/400 +TVimentin

(V9) Dako M725 1/2000 +MWDesmin Dako M760 1/500 NT

NT, no pretreatment; +T, trypsin pretreatment; +MW,microwave pretreatment.*Birmingham, UK; tKindly donated by David Lane, Universityof Dundee.

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Williams, Mepham, Wright

Table 4 Summary of immunostaining results afterincreasing fixation time in formalin

Antibody <5 h* 5 h 12 h 24 h 36 h 48 h

L26 +++ +++ +++ +++ +++ +++UCHL1 +++ +++ +++ ++ ++ +CD3 ++ ++ +++ +++ +++ +++Vimentin + +++ +++ +++ ++ ++Kappa - ++ +++ +++ +++ +++

*No trypsin digestion was used on sections fixed for < 5 hoursas even very short pretreatment times of less than 30 secondsseverely disrupted the tissue.

ing. The section cutting was staggeredfrom six months to one day so that all thesections could be stained at the same time.

IMMUNOSTAININGSections from each experiment were stainedwith a panel of five primary antibodies (table2). For the section preparation experimentsadditional tissues and antibodies were included(table 3). The primary antibodies were demon-strated using the StreptABC peroxidase (HRP)method as follows: sections were dewaxed andtaken through to 70% alcohol before treatingwith 0.5% hydrogen peroxide for 10 minutes toinhibit endogenous peroxidase. Sections werethen pretreated as required for each antibody(tables 2 and 3) and the trypsin time wasadjusted for each section to take into account

the type and period of fixation used. Sectionswere rinsed in Tris buffered saline (TBS) andincubated in primary antibody for the appro-priate time, that is, polyclonal immunoglobu-lins, 30 minutes at room temperature; mono-clonal and other polyclonal antibodies,overnight at 4°C. After a further rinse in TBS,sections were incubated in the relevant bioti-nylated second stage (Dako, High Wycombe,UK) for 30 minutes at room temperature, fol-lowed by incubation in StreptABC HRP com-plex (Dako) for the same period of time. TheHRP was visualised by reacting withdiaminobenzidine/H20, (Sigma Fast, Sigma,Poole, UK) for 10 minutes. After washing, thesections were counterstained in Harris haema-toxylin, blued in tap water, dehydrated throughalcohol to xylene, and mounted in DPX.

ASSESSMENT OF STAININGThe stained sections were examined independ-ently by two of the authors (BLM and JHW)without prior knowledge of the section treat-ment. The two sets of results were thencompared and any which differed by one gradeor more were re-examined by the two assessorstogether. If there were any further discrepan-cies the slides were re-assessed by the thirdauthor (DHW). The grades for recording

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AFigure 1 Sections of tonsilfixed in 10% NBF (A) and 10% formal acetic (B) stainedfor kappa light chain. Tissue fixed in 10% NBF shows staining offollicle centre cells, mantle cells, and plasma cells. Mantle cell staining is not seen in (B).

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Figure 2 Sections of tonsilfixedfor < 5 hours (A) and 48 hours (B) in 10% NBF and stained with UCHL1. Much stronger staining is seen with theshorter period offixation.

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Table S Summary of immunostaining results aftervarying thepH offormalin

Antibody pH 3.0 pH 5.0 pH 7.0 pH 9.0 pH 11.0

L26 +++ +++ +++ +++ +++UCHL1 +++ ++ ++ + +CD3 ++ +++ +++ +++ ++Vimentin +++ ++ ++ + +Kappa +++ +++ +++ +++ ++

intensity of specific staining were ,

> +++, excellent; +++, very good; +ate; +, weak; +/-, very weak; -, no s

USE OF MICROWAVE PRETREATMENTWhere weak staining was obtaiantibodies not usually requiring heathe staining was repeated using micrtreatment.

ResultsFIXATIONSeveral of the factors involved in thefixation were found to affect immunof some of the antibodies test(included the type, duration, and Ifixative. Of the types of fixatives teformal saline, 10% NBF (except UC

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10% zinc formalin (except CD3) gave the mostconsistent results overall and showed excellentantigen preservation. Of those recommendedfor immunocytochemistry, 10% formal acetic(fig 1), B5, and Bouin's fixative all showed poorantigen preservation. The use of B5 fixativeresulted in high levels of background staining.This was not improved by the use ofmicrowavepretreatment. Staining after Bouin's fixationwas weak, although the use of microwaving didenhance staining with L26 and UCHL1 butnot with CD3 and vimentin. Only weak immu-nostaining was obtained after using both thetraditionally used fixatives-10% NBFS and10% formalin in tap water-and the less com-monly used Carson's fixative.The period of fixation (table 4; figs 2 and 3)

and the pH of formalin (table 5; fig 4) also sig-nificantly affected the immunoreactivity ofsome of the antigens. UCHL1 and vimentinwere particularly susceptible to both longerfixation times and higher pH. In contrast, thedemonstration of both kappa and CD3 im-proved with increasing fixation times but wasless affected by changing the pH.The other fixation factors tested-delay

before fixation, volume of fixative, and the

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BFigure 3 Sections of tonsilfixedfor < 5 hours (A) and 48 hours (B) in 10% NBF and stained with kappa light chain. Tissuefixed for < 5 hours showspoor staining and appears overdigested, even though no trypsin pretreatment was used; 48 hours fixation results in clear staining offollicle centre cells,mantle cells, and plasma cells.

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Figure 4 Sections of tonsilfixed in 10% formalin atpH 3. 0 (A) andpH 11. 0 (B) and stained with vimentin. The staining in (B) is clearly muchweaker than in (A).

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Figure 5 Sections of tonsil processed at ambient temperature (A) and at 45°C (B) and stained with L26. The higher processing temperature givesstronger staining ofB cells.

Table 6 Summary of addition of methanol to formalin-had noimmunostaining results effect. Increasing the temperature of fixationfrom comparison ofprocessing temperatures was generally found to produce the same

results as increasing the time in fixative.Antibody Ambient 45'C

L26 +++ >+++ PROCESSINGUCHL1 +++ >+++ Increasing the processing temperature fromCD3 ++ +++ ambient to 45°C for all stages up to the waxVimentin ++ +++Kappa ++ ++ infiltration step (table 6; fig 5) and increasing

the duration of both the dehydration and waxinfiltration stages (table 7) improved theimmunostaining of most of the antibodiestested. None of the other factors investigated-the type of processor, type and quality ofreagent, use of vacuum, xylene contamination

Table 7 Summary of immunostainingfrom comparison of different processing times

Dehydration Wax infiltration

Total dehydration times Total wax infiltration times

Reduced Standard Extended Reduced Standard ExtendedAntibody (2 h) (7.5 h) (10 h) (2 h) (4 h) (8 h)

L26 ++ +++ >+++ + ++

UCHL1 +++ +++ +++ ++ ++ +++CD3 +++ +++ +++ ++ ++ ++

Vimentin +++ +++ +++ ++ ++ ++

Kappa ++ +++ +++ ++ ++ ++

Table 8 Summary of immunostaining results from a comparison of section dryingtemperatures and times

370C 60°C 600C 600C 600C HP HP HPAntibody ON I h 4h 8h ON 0.5h 2h 8h

TonsilL26 +++ ++ ++ ++ + + + +

UCHL1 +++ +++ ++ ++ ++ +1- +1- -

CD3* +++ +++ +++ +++ +++ +++ +++ +++

Vimentin ++ ++ ++ ++ ++ ++ ++ ++

Kappa +++ +++ +++ ++ ++ +++ +++ +++

MIB1 +++ +++ +++ +++ +++ +++ +++ +++

PC1o +++ +++ +++ +++ ++ + + _BCL-2 +++ +++ +++ +++ +++ +++ +++ +++

ColonaSMA +++ +++ +++ +++ +++ +++ +++ ++

CEA +++ +++ +++ +++ +++ +++ +++ +++

UterusVimentin

(V9) +++ +++ +++ +++ +++ +++ ++ +

Desmin +++ +++ ++ ++ +/- + +

Breast1D5 +++ +++ +++ +++ +++ +++ +++ +

ON, overnight; HP, hotplate at 700C.*Background staining increased with increasing temperature/time.

of formalin, length of clearing stage, and thenumber and position of the tissue in thecarrier-had any effect.

SECTION PREPARATIONThe temperature and duration of sectiondrying was found to affect immunoreactivity ofseveral of the antibodies tested (table 8; figs 6and 7). In contrast with section drying it wasfound that, with the exception of desmin, theimmunoreactivity of all antigens was unaf-fected in sections stored at room temperatureor at +40C for up to six months after cutting.

USE OF MICROWAVE PRETREATMENTThe use of microwave pretreatment was foundto reduce or eliminate most of the weak stain-ing occurring in this study: it improved immu-nostaining ofL26 and UCHL1 in Bouin's fixedtissue; it improved CD3 staining in tissue fixedfor less than five hours; and it significantlyimproved immunostaining with UCHL1 andL26 in sections dried at higher temperaturesand for longer periods.The only exceptions were: immunostaining

of all antibodies in B5 fixed tissue; CD3 andvimentin staining in Bouin's fixed tissue;vimentin staining in tissue fixed for less thanfive hours; and staining of most antibodies ifsections were dried on a hotplate for more than30 minutes.

DiscussionWe have shown that several factors in tissuepreparation can affect immunoreactivity, al-though the extent of this effect varies fromantibody to antibody. The findings suggest thatthere is no ideal tissue preparation schedule fordemonstrating all antigens, although varioussuggestions for improving immunocytochemi-cal staining can be made.The use of 10% NBF, 10% zinc formalin, or

10% formal saline is recommended, particu-larly for demonstrating kappa light chains onmantle and follicle centre cells.A fixation time of 12 hours in 10% NBF

showed optimum staining of all antigens, but assuch a defined time does not allow for variation

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of tissue type and composition and for the dayto day working of the laboratory, a fixation timeof between 12 and 24 hours is recommended.The pH of formalin affected different

antigens to varying degrees. A highly acidicformalin of pH 3.0 produced the best immu-nostaining but in our experience at the expenseof morphology. The use of formalin at pH 5.0is therefore recommended giving good mor-phology and immunoreactivity.

Generally the other factors involved infixation had little effect on the intensity ofimmunostaining provided that the recom-mended type, time, and pH of fixative wereused.Of the nine tissue processing factors investi-

gated, only two had any significant effect onimmunoreactivity. Increasing the temperatureof processing from ambient to 45°C, andlonger processing times for dehydration andwax infiltration were both found to improveimmunostaining. It is therefore recommendedthat wherever possible longer processing timesat higher temperatures should be used, and theuse of a short daytime schedule should beavoided for immunocytochemical studies. Wefound no effect of varying the other tissueprocessing factors, including the type ofprocessor, type and quality of reagents, time in

clearing agent, and use of vacuum, most ofwhich have been suggested as possible causesof poor processing. 1""17 19The conditions of section preparation have

mainly been ignored, but we have shown thatone of the fundamental steps of sectionpreparation-section drying-can have a sig-nificant effect on immunoreactivity. It isrecommended that sections, regardless of theantigen to be demonstrated, should be driedovernight at 37°C. If they are required morerapidly, then drying at 60°C for up to fourhours is acceptable, particularly if followed bymicrowave pretreatment. The use of a hotplate(70°C) for section drying should be avoided asthe staining with several of the antibodiestested was reduced and could not be improvedby microwave pretreatment. In contrast to thefindings of others,>"" this study has shown thatwith the exception of desmin (clone D33) allantibodies stained strongly in sections cut andstored for up to six months at either room tem-perature or +4°C.

It has been stated that heat pretreatmentenhances immunoreactivity and evens outirregular staining due to inadequate tissue fixa-tion or processing,2 34 and this is supported bythe findings of this study. It should be notedthat microwave pretreatment does not always

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Figure 6 Sections of tonsil stained with L26following section drying at 37°C overnight (A), 60°Cfor 4 hours (B), 60°C overnight (C), and on a

hotplate for 30 minutes (D). The results show the adverse effect of drying sections at high temperatures on a hotplate orfor long periods at 60°C.

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Figure 7 Sections of myometrium stainedfor desmin following section drying at 37°C overnight (A), 60°Cfor 4 hours (B), 60°C overnight (C), and on

a hotplate for 30 minutes (D). The adverse effect of high section drying temperatures is clearly shown.

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overcome the problem-for example, after theuse of high section drying temperatures. It istherefore suggested that "prevention is betterthan a cure", in that it is preferable to minimisethe chance of weak staining by using anoptimised fixation and processing schedulerather than producing sections which have tobe heat pretreated for immunostaining to beachieved.

Several of our findings are contrary to thosereported by others and this can be explained bythe variable response of different antigens tothe effects of tissue preparation. We thereforeconclude that there is no standard universaltissue preparation schedule for the optimaldemonstration of all antigens, although theimmunostaining of the majority of antigens canbe optimised by following the recommenda-tions given in this report.The results of this study have shown the

importance of resisting the pressure for shorterpreparation times for tissue requiring immuno-cytochemistry, as increased processing andsection drying times enhances immunoreactiv-ity. The results have also emphasised theimportance of including precise details oftissue preparation when reporting immuno-cytochemical findings rather than using thecommonly used term "routinely fixed andprocessed."

We thank Dako A/S, Denmark, for kindly sponsoring the studyand Dako UK for providing most of the antibodies.

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