Immunofluorescence on ultrathin resin sections相关交流-蚂蚁淘在线

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.

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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Immunofluorescence&nbsp;on&nbsp;ultrathin&nbsp;resin&nbsp;sections

Immunofluorescenceonultrathinresinsections

References:

Immunofluorescence on ultrathin resin sections