1.Deparaffinization/Rehydration
1.1.Heatslidesinanovenat65°Cfor1hour.
1.2.De-paraffinize/hydrateusingthefollowingseriesofwashes:twoXylenewashes（3mineach）,
followedbytwo100%ethanolrinses（3mineach）,followedby95%ethanol,70%ethanol,50%
ethanol,30%ethanol,followedbyTBSTwashfor3minonashaker.
2.AntigenRetrieval
ThisisrecommendedHeatInducedEpitopeRetrieval（HIER）usingDecloakingChamber/Pressure
Cooker.HotwaterbathorMicrowavewithtemperaturesensorcanbealsoused（protocolwouldvary
dependingonthemethodused）.
2.1.Add500mlofdH2OtoDecloaker/PressureCooker.
2.2.ImmerseslidesintostainingdishcontainingAntigenRetrievalSolution.Placestainingdishinto
decloakingchamber.
2.3.Programtorunfor30secondsat125°C,followedby10secondsat90°C.
2.4.Letitcooldowntoroomtemperature（10–20minutes）.
2.5.RemovesslidesandrinseinTBST.
2.6.ProceedtoStainingstep.
3.Staining
3.1.WashslideswithTBSTfor3minonashaker.
3.2.Inactivateendogenousperoxidasebycoveringtissuewith3%hydrogenperoxidefor5min.
3.3.WashslidesthreetimeswithTBST（3mineachonashaker）.
3.4.Blockslideswiththeblockingsolutionfor1hour.
3.5.Diluteprimaryantibodyinprimaryantibodydiluentperrecommendationondatasheet.
3.6.Applyprimaryantibodytoeachsectionandincubateovernightinthehumidifiedchamber（4oC）.
3.7.WashslidesthreetimeswithTBST（3mineachonashaker）.
3.8.ApplytoeachsectionsecondaryHRP-conjugatedanti-rabbitantibodydilutedintheblocking
solutionpermanufacturer’srecommendation;incubatefor30minatroomtemperature.
3.9.WashslidesthreetimeswithTBST（5mineachonashaker）.
3.10.AddfreshlypreparedDABsubstratetothesectionsandincubateuntilstaindevelops（generally1
min）.
3.11.Rinsesectionswithwater.
3.12.CounterstainwithHematoxylin（generally10seconds）.
3.13.Rinsesectionswithwater.
3.14.Dehydratesamplesusingtwowasheswith100%Ethanol（3mineach）,followedbytworinses
withXylene（3mineach）.
3.15.Mountcoverslipsonslidesusingpermanentmountingmedium
