• ImmunofluorescenceTechnique(SpectorLab)protocolforimmunofluorescenceoncells
• ImmunofluorescenceProtocol(WalterSteffen)
  • Preparationofcoverglasses
  • Cellculture
  • Fixationprotocol
    • Methanolfixation
    • Formaldehydefixation
    • Paraformaldehyde/glutaraldehydefixation
    • EGSfixation
    • FixationoftheCytoskeleton
  • Immunofluorescenceprotocol
  • Doubleimmunofluorescence
  • ProteinA-proteinG
  • Avidin-biotin
• Immunofluorescence(CorbettLab)Yeastprotocol
• ImmunfluoresenceonChamberSlides(BowtellLab)
• Immunofluorescence(withMethanol)(D.AmbergLab)ThisistheprotocoltousewiththeBotsteinanti-actinrabbitantibodies
• Immunofluorescence(withoutmethanol)(D.Amberg)
• ASemi-PermenantMountingMediumforImmunofluorescenceMicroscopy(HouseEarInstitute)ArecipeforMoviol,aninexpensivemountingmediumthatiseasytoprepare.
• InSituHybridizationofmRNAandImmunofluorescence(CorbettLab)Yeastprotocol
• ImmunofluorescenceStainingandFlowCytometryofIntracellularCytokines(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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