贴壁细胞的免疫荧光染色方法(ProtocolofImmunofluorescence(IF)onat

贴壁细胞的免疫荧光染色方法（ProtocolofImmunofluorescence(IF)onattachedcells）Materials:1.PBSsolution2.4%Paraformaldehyde(PFAfixative):Dissolve4gparaformaldehydein100mlPBSsolution,stirat70℃todissolve;3.PBS-Tsolution:(0.1%TritonX-100inPBSsolution)4.PBS-Bblockingsolution:(4%BSAinPBSsolution)5.Primaryantibody:DilutewithPBS-Bsolution,dilutionfactorsshouldrefertomanual,or,testfrom1:50~200,shouldbemoreconcentratethanapplicationinWesternblot;6.Secondaryantibody:DilutewithPBS-Bsolution,dilutionfactorsshouldrefertomanualProcedure:1.Culturedcells,letitattachtothecoverslipsin6-wellplate;2.Removemedium,rinsewithPBStwice;3.Add2ml4%paraformaldehyde,incubateatroomtemperaturefor20minutes;4.Rinsewith2mlPBSthreetimes,rinsefor5minuteseverytime;5.Permeabilizecellswith2mlPBS-Tsolutionat4℃for10minutes;6.RemovePBS-Tsolution,rinsecellswithPBSfor5minutesatroomtemperature;7.Blocknon-specificinteractionwithPBS-Bsolutionat37℃for30minutes;8.Addprimaryantibodysolution,incubateat4℃overnight;9.Removeprimaryantibodysolution,washwithPBSfor5minutes;10.Addsecondaryantibodysolution,incubateatroomtemperaturefor1hour;11.WashwithPBSthreetimes,5minuteseach;12.Addanti-fadeDAPIsolutionifneeded;13.Observation.