staining-intracellular-antigens-for-flow-cytometry(1).pdfVIP免费

FlowCytometry–BestProtocols®Page1of9StainingIntracellularAntigensforFlowCytometryResearchUseOnlyRevised6-24-2013ProvidedasacourtesybyeBioscience,anAffymetrixCompany•Copyright©2000-2013eBioscience,Inc.Tel:888.999.1371or858.642.2058•Fax:858.642.2046•www.ebioscience.com•info@ebioscience.comProtocolA:Two-stepprotocol:intracellular(cytoplasmic)proteinsProtocolB:One-stepprotocol:intracellular(nuclear)proteinsProtocolC:Two-stepprotocol:Fixation/MethanolIntroductionAmodificationofthebasicimmunofluorescentstainingandflowcytometricanalysisprotocolcanbeusedforthesimultaneousanalysisofsurfacemoleculesandintracellularantigensatthesingle-celllevelbyflowcytometry.Typically,cellsarefixedwithformaldehydetostabilizethecellmembrane,andthenpermeabilizedwithdetergentoralcoholtoallowantibodiesagainstintracellularantigensaccesstostainintracellularly.Whenstainingproteinsinsidethecell,itisimportanttoconsidertheirlocationasthismaydictatetheprotocolandbuffersystemthatwillperformoptimally.Forexample,nuclearproteinsandmanysecretedproteinsworkwellwiththeeBioscienceFoxp3/TranscriptionFactorStainingBufferSet(eBioscienceCat.No.00-5523),whilesecretedproteinssuchascytokinesandchemokinesworkwellwiththeIntracellularFixationandPermeabilizationBufferSet(eBioscienceCat.No.88-8824).Lastly,thereareseveralphosphorylatedsignalingproteinsthatmaynotworkinthetwopreviously-mentionedbuffersystemsbutwillworkwiththeFixation/MethanolProtocol.Informationaboutperformanceandpreferredbuffersisnotedonthespecificproduct’sTechnicalDataSheet.PleasecontactTechnicalSupport(tech@ebioscience.com)formoreinformation.GeneralNotes1.Foroptimalperformanceoffluorochrome-conjugatedantibodies,storevialsat4°Cinthedark.Donotfreeze.2.Priortouse,quicklyspintheantibodyvialtorecoverthemaximumvolume.Wedonotrecommendvortexingtheantibodyvial.3.Exceptwherenotedintheprotocol,allstainingshouldbedoneoniceorat4°Cwithminimalexposuretolight.4.Thefixationandpermeabilizationstepsthatarerequiredforthedetectionofintracellularantigensmayal...