protozoan staining protocol

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1、AECIMMUNOSTAININGPROCEDURERevised01-10-111.DeparaffinizeslidesinoldxyleneIfor20min.DiscardxyleneI.PourxyleneIIintoxyleneIcontainer.Soak5min.2.PourfreshxyleneintoxyleneII.Soakslidesinfreshxylenefor5min.3.Soakin100%ethanolIfor5min.DiscardethanolI.PourethanolIIintoethanolIcontainer.Soak5min.Po

2、urfresh100%ethanolintoethanolIIcontainer.Soakanother5min.4.Soakin95%ethanolfor5min.5.Quenchendogenousperoxidasein3%H2O2inmethanolfor15min.(20mlhydrogenperoxideand180mlmethanol).6.Soakin80%ethanolfor5min.7.Soakin70%ethanolfor5min.8.Soakin50%ethanolfor5min.9.Soakinsalinefor5min.10.Soakinwarms

3、aline(37C)for5min.11.Pepsindigestion:incubateslidesin0.4%pepsinin0.01NHClfor15min.at37C.(0.8gmpepsin,0.333ml6NHCl,200mlsaline,prewarmedto37Cfor30min).12.Soakin0.75%BRIJ-PBSfor5min.twice(2.5ml30%BRIJ35perliterPBS).13.Blocknonspecificbindingwith0.5%NacaseinateinBRIJ-PBSfor10min.intheslidemoat

4、.14.Drainandwithoutrinsingapplytheproperlydilutedprimaryantibodytothetissuesfor30min.at37C.15.SoakslidesinBRIJ-PBSfor5min.twice.16.TreattissueswithDakoEnVisionRabbitperoxidase(catalog#K4003)solutionat37Cfor30min.17.SoakslidesinBRIJ-PBSfor5min.twice.18.ApplyDakoAECsubstratechromogen(catalog#

5、K3469)solutiontoslidesat37Cfor15min.19.Collectchromogeninhazardwastebottle,andsoakslidesinBRIJ-PBSfor5min.20.Rinseslidesinrunningtapwaterfor2min.Rinseinde-ionizedwaterfor2min.twice.21.CounterstainwithMayer’sHematoxylinfor1min.22.Washslidesinrunningtapwaterfor1min.23.SoakslidesinScott’stapwa

6、tersubstitute(MgSO4.7H2O20gm,NaHCO32gm,water1liter)for1min.24.Soakinrunningtapwaterfor1min.Soakinde-ionizedwaterfor2min.twice.25.ApplyCrystalMountandincubateslidesovernightatroomtemperature.26.ApplyPermountandcoverslip,ifdesired.Staining.wpd