Immunofluorescenceonultrathinresinsections
Thisprotocolistakenfromthe1999FEBScoursemanualandwascontributedbyHeinzSchwarz.
| Generalremarks:Ultrathinmethacrylate-(e.g.LowicrylHM20,K4M,MonosteppolarandnonpolarorLR-White)orepoxy(Epon/Araldite,Spurr)sectionsaretransferredwithalooponpoly-L-lysinecoated,roundcoverslips.Residualwaterisdrainedwithfilterpaperalongtheoutsideoftheloop. Note:air-driedresinsectionscanbestoredformonthspriortolabelling. |
Procedure:- Encirclesectionswithawaterrepellentsiliconpen(e.g.PAP-PenfromElectronMicroscopySciences,Ft.Washington,PAorSciencesServices,Munich).
- Incubatesectionswithblockingbuffer,e.g.PBG(0.2%gelatin,0.5%BSAinPBSorTRIS)or1%milkpowderinPBSfor10min.
- Removeblockingbuffer,add25µloftheprimaryantibodysolutionpercoverslip(withafinalconcentrationintherangeof1-5µgspecificIgG/ml)andincubatefor30-60min.
- Wash5timeswithbufferandincubatewithFluorochrome-labelledsecondantibodies,similartotheprimaryantibodystainingconditions.sWash5timeswithbufferandcounterstainnucleiwithDAPI,Hoechstdyeorpropidiumiodide(0.4-0.1µg/mlinH2O)for5min.
- Afterafinalwashwithbuffermountcoverslipsonglassslidesusingasmalldropofmountingmedium(likeElvanolorMoviol4.88)forsemipermanentembedding.Theadditionofanti-fADIngagentslikeDABCO(25-100mg/ml),Paraphenylenediamine(1mg/ml)orn-propylgallate(10mg/ml)isstronglyrecommended.
- Useoilimmersionobjectivestoexamine.CAVEAT:Allsolutionsshouldbecentrifugedbeforeusefor2min.at10,000rpm.Avoidair-dryingduringallincubationsteps.
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References:Immunofluorescenceonultrathinresinsections: - AlbrechtU,SeulbergerH,SchwarzH,andRisauW.(1990)BrainRes.535,49-61.
- SchwarzH,HohenbergH,andHumbelBM.(1993)Immuno-GoldElectronMicroscopyinVirusDiagnosisandResearch(CRCPress,BocaRaton),pp349-376.
- SchwarzH,Müller-SchmidA,andHoffmannW.(1993)CellTissueRes.273,417-425.
- SchwarzH.(1994)ElectronMicroscopy1994ICEM13-Paris(JouffreyB,andColliexC,eds.),LesEditionsdePhysique,LesUlis,France,Vol.3,255-256.
- FialkaI,SchwarzH,ReichmannE,BusslingerM,andBeugH.(1996)J.CellBiol.132,1115-1132.
- KurthT,SchwarzH,SchneiderS,andHausenP.(1996)CellTissueRes.286,1-12.
Semi-permanentmountingmediumcontainingpolyvinylalcohols(Elvanol/Moviol): - RodriguezJ,andDeinhardtF.(1960)Virology12,316.
- LennetteOA.(1978)Am.J.Clin.Path.69,647-648.
Antifadingagents: - 1,4-Diazobicyclooctane(DABCO):
- JohnsonGDetal,1982.J.Immunol.Meth.55,231-242.LangangerG,DeMeyJ,andAdamH.(1983)Mikroskopie40,237-241.
- Paraphenylendiamine:
- JohnsonGD,andAraujoGMDCN.(1981)J.Immunol.Meth.43,349-350.
- n-Propylgallate:
- GilohH,andSedatJW.(1982)Science217,1252-1255.
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