ImmunohistochemistyEnzymatic Protocol

Overview

R&DSystemsprovidesmonoclonal,polyclonalandbiotinylatedantibodiesforimmunohistochemicaluse.ThefollowingprotocolhasbeendevelopedandoptimizedbyR&DSystems""ImmunohistochemicalLaboratory.R&DSystems""antigenaffinity-purifiedpolyclonalandmonoclonalantibodieshavebeenusedtostainfrozencellsandtissues,aswellasparaffin-embeddedtissues.Immunohistochemistryprotocolsmayrequiremodificationdependingonthetypeoftissueused.Eachinvestigatorshoulddeterminetheoptimalconditionsandworkingdilutionsofantibodies.IfusingR&DSystems""primaryantibodies,refertothespecificproductinserttoobtainanapproximateworkingdilution.Forallotherreagents,followthemanufacturer""sinstructions.ForResearchUseOnly.

Materials

PrimaryAntibodies

Unlabeledorbiotinylatedantigenaffinity-purifiedpolyclonalantibodies(R&DSystems""AForBAFseries)orselectedunlabeledorbiotinylatedmonoclonalantibodies(R&DSystems""MABorBAMseries)

Cell&TissueStainingKits-Enzymatic/Chromogenic

• AgainstGoatPrimaryAntibodies:
  • HRP-DAB(Catalog#CTS008)
  • HRP-AEC(Catalog#CTS009)
• AgainstMousePrimaryAntibodies:
  • HRP-DAB(Catalog#CTS002)
  • HRP-AEC(Catalog#CTS003)
• AgainstRabbitPrimaryAntibodies:
  • HRP-DAB(Catalog#CTS005)
  • HRP-AEC(Catalog#CTS006)
• AgainstRatPrimaryAntibodies:
  • HRP-DAB(Catalog#CTS017)
  • HRP-AEC(Catalog#CTS018)

BuffersandAdditionalSupplies

• Fixative:85mMNa2HPO4,75mMKH2P04,4formaldehyde(SigmaCatalog#P6148)and14(v/v)saturatedpicricacid(SigmaCatalog#925-40),pH6.9.Picricacidisoptional.
• SucroseSolution:130mMNa2HPO4,30mMKH2PO4,10(w/v)sucrose(SigmaCatalog#S7903),0.01sodiumazide(SigmaCatalog#S2002)and0.03Bacitracin(SigmaCatalog#B-0125),pH7.2
• PBS:50mMNa2HPO4and140mMNaCl,pH7.2
• IncubationBuffer:1bovineserumalbumin(SigmaCatalog#A2153),1normaldonkeyserum(SigmaCatalog#D9663),0.3TritonX-100(SigmaCatalog#T9284)and0.01sodiumazide(SigmaCatalog#S2002)inPBS
• AqueousMountingMedium(Catalog#CTS011)
• DABEnhancer(Catalog#CTS010)
• AntigenRetrievalReagents(Catalog#CTS013,CTS014,CTS015orCTS016)

Note:Equivalentchemicalsandreagentsmaybesubstitutedforthoselistedabove.

SamplePreparation(FrozenTissues)

Thevastmajorityofimmunohistochemicalproceduresemployacellortissuefixationstepusingformaldehydeorothercross-linkingfixativespriortoincubationwithprimaryantibody.Fixationisrequiredtoretaintissuemorphologyandpreventdegradationoftissueantigens.Fixationmaybeperformedeitherbyimmersingdissectedpiecesoftissue(e.g.humanbiopsies)intothefixative,orbyvascularperfusion(e.g.laboratoryanimalssuchasmice,rats,guineapigs,etc.).Itisveryimportanttooptimizefixingconditionssinceunder-orover-fixationmayreduceorabolishtissueimmunoreactivity.Theeasiestwaytocorrectunder-fixationistopost-fixtissuesectionsontheslidebeforestartingimmunohistochemicalstaining.Torecoverantigensinover-fixedtissues,eitherprotease-inducedepitoperetrieval(PIER)orheat-inducedepitoperetrieval(HIER)techniquesarerecommended.HIERcanbeperformedusingamicrowaveoven,pressurecooker,vegetablesteamer,autoclaveorwaterbath.Aftertissuesarefixed,theymayeitherbeembeddedintoparaffinorcoveredwithOCTcompoundandfrozenforfurthersectioning.Paraffin-embeddedtissuesarecutusingamicrotomeatroomtemperature,whereasfrozentissuesarecutusingacryostatattemperaturesbelow0°C.Antigenimmunoreactivitywasfoundtobebetterpreservedinfrozenratherthanparaffin-embeddedtissues.1,2

References

1. Larsson,L.-I.(1988)Immunocytochemistry:TheoryandPractice,CRCPress,BocaRaton,Florida.
2. Frost,A.etal.(2000)Methodsofantigenrecoveryvaryintheirusefulnessinunmaskingspecificantigensinimmunohistochemistry,Appl.Immunohistochem.Mol.Morphol.8:236.

TissueFixationandMounting-CryostatSections

1. Fixthetissuebyvascularperfusionwith500-700mLofFixative.
2. Perfusetheanimalwith400mLofSucroseSolution.
3. Dissectthetissue,mountinOCTandfreezeat-20to-80°C.
4. Cut5-15µmthicktissuesectionsusingacryostat.
5. Thaw-mountthesectionsontogel-coatedslides.RefertotheSupportProtocolsectiontofollowforinstructionsonhowtopreparegel-coatedslides.
6. Drytheslidesfor30minutesonaslidewarmerat37°C.Slidescontainingcryostatsectionscanbestoredat-20to-70°Cforupto12months.

TissueFixationandMounting-Paraffin-embeddedSections

1. Fixthetissuebyvascularperfusionwith500-700mLofFixative.
2. Dissectthetissue.
3. Immersethetissuein70ethanolthreetimesfor30minuteseachatroomtemperature.
4. Immersethetissuein90ethanoltwotimesfor30minuteseachatroomtemperature.
5. Immersethetissuein100ethanolthreetimesfor30minuteseachatroomtemperature.
6. Immersethetissueintoluenethreetimesfor20minuteseachatroomtemperature.
7. Embedthetissueinparaffin(Paraplast,FisherScientific)twotimesfor60minuteseachat58°C.Alternatively,tissuescanbeembeddedintoparaffinusingspecializedautomatedtissueprocessingsystems.
8. Cut5-15µmthicktissuesectionsusingarotarymicrotome.
9. Floatthesectionsina56°Cwaterbath.
10. Mountthesectionsontohistologicalslides.
11. Drytheslidesovernightatroomtemperature.Slidescontainingparaffin-embeddedsectionscanbestoredatroomtemperature.

WhenitisnotpossIBLetofixtissuebyperfusion,dissectedtissuemaybefixedbyimmersingthetissueintoa10formalinsolutionfor4-8hoursatroomtemperature.Itiscommonlyacceptedthatthevolumeoffixativeshouldbe50timesgreaterthanthesizeoftheimmersedtissue.Avoidfixingthetissueforgreaterthan24hourssincetissueantigensmayeitherbedestroyedormasked(A.C.Cuello,ed.,1993,Immunohistochemistry:MethodsintheNeurosciences,Vol.14;IBROHandbookSeries,JohnWiley&Sons,NewYork).

SupportProtocols

GelCoatingSolution:

1. Dissolve5ggel[typeA,175bloomfromporcine(SigmaCatalog#G2625)]in1Lwater,whileheating.Donotallowthetemperaturetoexceed45°C.
2. Add0.5gchromiumpotassiumsulfate[CrK(SO4)2.12H2O]anddissolvecompletely.Storeat4°C.Thissolutioncanbere-used3-4timesandisstablefor1-2monthswhenstoredat4°C.

Gel-coatedSlides:

1. HeattheGelCoatingSolutionto40-44°C.
2. Filterthesolutionusingcoarsefilterpaper.
3. Pourthesolutionintoalargestainingdish.
4. Removebubblesfromthesurfaceofthesolution.
5. Brieflysubmergearackcontaininghistologicalslidesintothesolution.
6. Removetherackofslidesfromthestainingdish.
7. Placetherackontoapapertowelandcovertheslideswithpapertowelstoavoidcontaminationwithdust.
8. Allowtheslidestodryatroomtemperature.Dryslidescanbestoredfor1yearat-20°C.

Antigen-retrievalProtocolThisprotocolisbasedonusingR&DSystems""AntigenRetrievalReagents(Catalog#CTS013,CTS014,CTS015orCTS016).Note:theantigen-retrievalcapacityofeachAntigenRetrievalReagentdependsonsamplepreparation,antigenstructure,incubationtime(upto30minutes)andtemperature(90-100°C).Eachinvestigatorshoulddetermineoptimalconditions.

1. Dilutethe10XAntigenRetrievalReagent10-fold,usingdeionizedwater,tomaketheRetrievalSolution.
2. HeattheRetrievalSolutionto92-95°C.ThismaybeaccomplishedbyplacingapolypropyleneCoplinstainingjarfilledwithRetrievalSolutionintoawaterbath.Note:heatingmaycrackglassstainingdishes.
3. InserttheslidescontainingtissuesintotheheatedRetrievalSolutionandincubate2-10minutes.Note:cryostatsectionsaremoresusceptibletothedamagingeffectsoftheRetrievalSolutionthanparaffin-embeddedtissues.Toavoidtissuedamage,itmaybenecessarytoshortentheincubationtimeto2-5minutesforcryostatsections.
4. PlacetheCoplinjarcontainingtheRetrievalSolutionandslidesonalabbenchandallowtocoolfor5-10minutesatroomtemperature.
5. RinsetheslideswithdistilledwaterfollowedbyPBS.Note:tissuesmaybecomelooseaftertheretrievalprocedure,avoidvigorousrinsingtopreventdetachmentofthetissuesfromtheslides.Proceedtostep5oftheTissueStainingProtocol.