Sourceofunwantedstaining,besidespoorknowledgeoftheantibodyreactivityandmalice,isdueto:

EndogenousenzymesorFluorochromes.

Endogenousbiotin.

Endogenousantibodybindingactivity(Fcreceptors).

Crossreactivityofthesecondaryreagentswithendogenousproteins.

BlockingofendogenousenzymesEndogenousenzymessuchasAlkPhos,AcPhosandesterasesaredestroyedbyboilingRef,evenashorttimeat100C.Peroxidaseisnot.Blockingofendogenousperoxidaseisdonebypreincubatingtheslidesin

3H2O21SodiumAzidePBS30minutesseveralwashes

Thismixtureismoreeffectivethan1.5H2O2inabsolutemethanolRef.Methanolincubationisalsoafixationstepthatmayaffectyourstaining.BlockingofendogenousAlkalinePhosphataseisdonebyexploitingthedifferentialsensitivityofcalfintestinalAP,usedasareporter,andleukocyteAP.Thislatterisinhibited,whiletheformerisnotbyLevamisole1mM,whichshouldbepresentinthedevelopmentbuffer.

Blockingofendogenousfluorochromes.BlockingofendogenousfluorochromesisimpossIBLe.OnemaychoosefluorochromesemittingintheUVrangeofspectrum,whereendogenousautofluorescenceoftissueisminimal.

BlockingendogenousbiotinCanbedonewithcommerciallyavailablekitsorbybuyingtheisolatedcomponentsofthekits,freebiotinandfreeavidin.Canalsobedonewithsimplerreagentsasbelow.

1-WashtwiceinTBS0.05MpH7.5,towhich0.01Tween20hasbeenadded(TBS-T).

2-BrieflyblottheslideswithoutlettingthemdryandthenapplyeggwhiteinPBS-BSA-NaN3asablockingagent(oneeggwhitein100mlPBS+NaN3)(MillerRTetal,ApplImmunohistochemistry5(1):63-66,1997).Incubatefor10min.minimum.(UsetheyolkasdirectedinCucinait.com)

NOTE:thispassagemaybeomitted,howeversubsequentdoublestainingwithabiotinbasedmethodmayneedendogenousbiotinblocking.

3-WashonceinTBS-T.

4-Incubatewithskimmilk5inTBS-Tfor10-30min.(MillerRTetal,Appl.Immunohist&MolecularMorphology,71(1):63-65,1999)

NOTE:skimmilkmaycontainweakproteaseswhichwillbeusefultofurtherunmaskantigens,butmayreduceimmunostainingbyactingonprimaryAbs.

5-WashonceinTBS-T(residualmilkcancontributetoblocking).

BlockingofendogenousFcblocking.SpecimensnotparaffinembedddedmayhavesignificantFcbindingactivitybymacrophages,Bcells,Tcellsandothercelltypes.ByexploitingthepreferentialAvidityofFcreceptorforhuman>mouseIg>rabbit>swine>goatimmunoglubulins,onemayuseablockingofthereceptorswithareagentwhichwillnotinterferewiththesecondaryreagentsandwithwhichthesecondaryantibodiescanbeabsorbed(1serumadded).

Blockingofcrossreactiveantigensinthetissue.Typicalexampleisstainingmousemonoclonalsinmousetissue,whereendogenousimmunoglobulinswillbespecificallydetectedbytheantibodyaimedattheexogenousantibodyused.ThistopicisdiscussedintheMouse-on-MouseeasyIHCsectionofthismanual.Hapten-conjugationoftheprimaryantibody(FITC,Pe,Biotin,Deoxygenin)willsolvetheproblem.

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