免疫荧光
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- ImmunofluorescenceTechnique(SpectorLab)protocolforimmunofluorescenceoncells
- ImmunofluorescenceProtocol(WalterSteffen)
- Preparationofcoverglasses
- Cellculture
- Fixationprotocol
- Methanolfixation
- Formaldehydefixation
- Paraformaldehyde/glutaraldehydefixation
- EGSfixation
- FixationoftheCytoskeleton
ImmunofluorescenceprotocolDoubleimmunofluorescenceProteinA-proteinGAvidin-biotinImmunofluorescence(CorbettLab)YeastprotocolImmunfluoresenceonChamberSlides(BowtellLab)Immunofluorescence(withMethanol)(D.AmbergLab)ThisistheprotocoltousewiththeBotsteinanti-actinrabbitantibodiesImmunofluorescence(withoutmethanol)(D.Amberg)ASemi-PermenantMountingMediumforImmunofluorescenceMicroscopy(HouseEarInstitute)ArecipeforMoviol,aninexpensivemountingmediumthatiseasytoprepare.InSituHybridizationofmRNAandImmunofluorescence(CorbettLab)YeastprotocolImmunofluorescenceStainingandFlowCytometryofIntracellularCytokines(eBioscience)Amodificationofthebasicimmunofluorescencestainingandflowcytometricanalysisprotocolcanbeusedforthesimultaneousanalysisofsurfacemoleculesandintracellularcytokinesatsingle-celllevel.Inthisprotocol,cellsarefirstactivatedinvitro,stainedforsurfaceantigens,asintheregularstainingprotocol,thenfixedandpermeABIlizedtoallowforanti-cytokineantibodiestostainintracellularly.FluorescenceMountingMedium(Antifade)(SpectorLab)Large-scaleImmunofluorescence(YGAC)Thisprotocoldescribesmethodforpreparingcellsforimmunofluorescence,inwhichallincubationsandwashesareperformedinmicrotiterdishes.Twodishescanbeprocessedsimultaneously.Thelaststepistotransfereachsampletoawellonapolylysinecoated8-wellslideforvisualization.Theprotocolcanalsoeasilybeadaptedforpreparingcellsforimmunofluorescenceinmicrofugetubes.ImmunocytologyusingtheHAEpitope(YGAC)ThemultiplecopiesoftheHAepitopepresentintheHATtagcanbedetectedbythemousemonoclonalantibodies12CA5(Boehringer)and16B12...Stainingforsurfacereceptorsbyfeedingantibody(VonZastrowlab)Thisprotocolsallowstheuseoffluorescencemicroscopytovisualizeepitope-taggedreceptorsexpressedbystableadherentcelllines.Incontrasttothepermeabalizedcellprotocol,thisprotocolwhereantibodyis"fed"toliveunpermeabalizedcellswillonlyvisualizereceptorsthatstartedoutonthesurfaceofthecell.·Stainingfortotalreceptor(VonZastrowlab)Thisprotocolwillvisualizebothinternalizedreceptorsthatwereonceontheplasmamembraneandreceptorsinthebiosyntheticpathway.ThisprotocolwasspecificallywrittenforvisualizingagoNIST-inducedinternalizationofreceptors.
·AntibodyCleanup(YGAC)Inordertodecreasetheamountofnonspecificstaining,itisoftennecessarytopreabsorbprimaryandsecondaryantibodiestoyeastcellslackingtheantigenpriortouse.
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