Preface:

Youshouldnotco-staininIHCantigenslocatedonthesamestructure(nucleus,membrane,cytoplasm).Thatbecausethesuccessofthisdoublestainingprocedureisbasedonthecompletedevelopmentofthefirststain,whichshouldmaskthestructurestainedfirst.Thereforeyoushouldbeabletousetwoantibodiesraisedinthesamespecies.Howeverbetterresultsareobtainediftwodifferentspeciesareused.CarefulcheckpossIBLecrossreactivtyofyoursecondaryantibodiesbystaininginsimpleIHCtherelevantheterologousprimaryantibodies.E.g.manygoatantimouseIgG₁heavychaindostainratIgG_2aandIgG_2b.

• Firststain
• HRPDevelopingsolution
• Secondstain
• APDevelopingsolution

Applythecorrectprocedure(fixationorantigenretrieval)tothesections.Applytheblockingprocedure.Beawarenottouseforblockinganycomponentwhichmaybedetectedbyanysecondaryantibodyyouplantouse.

Firststain:Indirectimmunohistochemistrywithavidin-biotinperoxidase(e.grabbitantiantigenX,goatanti-rabbitbiotin,avidinHRP)

1-WashtwiceinTBS0.05MpH7.5,towhich0.01%Tween20hasbeenadded.2-Brieflyblottheslideswithoutlettingthemdryandthenapply3%humanorpigserumasablockingagent(healthhazard!).3-incubatewiththeblockingfor10min.Ifoneofyourantibodyisbiotin-conjugated,youneedatthispointtodoendogenousbiotinblocking.4-blottheslideswithoutwashingandapplytheprimaryantibody,inamoistchamber,atRTfor1-18hr.5-WashtwiceinTBS0.05MpH7.5+0.01%Tween20.6-addthebiotin-conjugatedsecondaryantibody(50to100µl)andincubatefor45min.Thesecondaryantibodyshouldbeabsorbedagainsthumanserum;ifnotadd1%humanserumbeforeuse.Mostreagentsareusedat1:100or1:200dilution.7-WashtwiceinTBS0.05MpH7.5+0.01%Tween20.7bis-blockendogenousperoxidasebyincubatingin0.1%NaN₃and0.3%H₂O₂for30min.Washthrice.8-addtheHRP-conjugatedavidin(50to100µl,dilution1:300-500innTBS-Tween)andincubatefor20min.Becarefulnottodilutetheavidininbiotin-containingmedium.9-WashthriceinTBS0.05MpH7.5+0.01%Tween20.]10-add50mlofthedevelopingsolution(seebelow).Protectfromdirectlight.11-after5min,checkthestaininginyourpositiveandnegativecontrols.12-checkthestaininguntilcomplete,densestainingisobtained,butbackgroundisstilllow.13-whenstainingiscomplete,washthoroughlyintapwater.14-transfertoTBS0.05MpH7.5+0.01%Tween20.

HRPDevelopingsolution:For50mldevelopingsolutionaddinorder:

• Aminoethylcarbazole(20mgtablets,SigmaA-6926,dissolvedin2.5mlNN-DMformamide)
• 50mlacetatebufferpH5.5(52.5mlof0.1Maceticacidsolution+196.5mlofa0.1MNaacetatesolution,bringto500ml)
• 25µlH₂O₂30%.

ShakewellFilterwitha45µmfilter(optional).Keepawayfromdirectlight,usewithin5min.

Secondstain:Doubleindirectimmunohistochemistry(e.grabbitantiantigenX,goatanti-rabbitAP,rabbitanti-goatAP)

Asecondblockingstepisomitted,becauseblockingisduetothefirststain.Onecanbrieflyboiltheslidestore-retrieveantigensandquenchtheprimarylayer.(bringtoaboilinEDTAbufferandletcool).

1-applythe2ndprimaryantibody,inamoistchamber,atRTfor1-18hr.2-WashtwiceinTBS0.05MpH7.5+0.01%Tween20.3-addtheAPconjugatedsecondaryantibody(50to100µl)andincubatefor45min.Thesecondaryantibodyshouldbeabsorbedagainsthumanserum;ifnotadd1%humanserumbeforeuse.SBAGoatantimouseAPorGoatantiRabbitAPcanbeused1:200inTBS-BSANaN₃.4-WashthriceinTBS0.05MpH7.5+0.01%Tween20.5-addtheAPconjugatedtertiaryantibody(50to100µl)andincubatefor15min.Thetertiaryantibodyshouldbeabsorbedagainsthumanserum;ifnotadd1%humanserumbeforeuse.SBAGoatantimouseAPorGoatantiRabbitAPcanbeused1:200inTBS-BSANaN₃.6-WashthriceinTBS0.05MpH7.5+0.01%Tween20.7-add50mlofthedevelopingsolution(seebelow).Protectfromdirectlight.8-after5min,checkthestaininginyourpositiveandnegativecontrols.9-checkthestainingat10-15mininterval.10-whenstainingiscomplete(usually<1hr),washthoroughlyintapwater.11-preferablypostfixinformalinfor4-5hrsbeforemountinginwatersolublemountingmedium(glycerolgelatin).

Donotcounterstain,unlessyoucanaffordaverygentlehematoxilynhueinthenuclei.

APDevelopingsolution:For50mldevelopingsolutionaddinorder:

• 50mlTris-Hcl0.1MpH9.2(exact!)(1:10fromastocksolution1M)
• Levamisole1mM(12mg)
• 20mgNaphtolAsBIphosphate(stocksolution40mg/mlinNN-DMformamide,anhydrous,keptat-20°C)
• 10mgFastBlueBBDiazoniumsalt(SigmaF3378)

ShakewellFilterwitha45µmfilter.Keepawayfromdirectlight,usewithin5min.

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