Freeze Substitution相关交流-蚂蚁淘在线

FreezeSubstitution

Howtopreparesamplesusingfreeze-substitutionwithoutanexpensivemachine.

Thismethodhasbeenusedsuccessfullyinourlaboratorysince1994.TheprotocolassumesalluserswillbefamiliarwithroutineTEMpreparationmethods.AnimportantapplicationofthismethodwasmadebyDr.A.WhitneywhousedittoexamineMDCKcellsgrownonfilters.

AldehydeFixation: Fixcellsortissuesinbufferedaldehydesolution.Weuseeither4%formaldehyde(madeupfromparaformaldehyde)oramixtureof4%formaldehydeand0.2%gluteraldeyhde,bufferedin100mMphosphatebuffer.Fixationtimesvarybutroutinelyweuse1hr.Wholeorgansarebestfixedbyperfusiingthewithfixative.
Cryoprotection: Incubatefixedmaterialin2.3Msucrose.Thiscryoprotectionmethodisalsousedformaterialthatistobefrozenforcryosectioning.Smallpiecesofthematerialareleftfor1hourtoinfiltrate.
Freezing: Mountthesmall,cryoprotectedsamplepiecesontometalpinsandimmerseinliquidnitrogen.Thepinsareusefulformanipulatingthefrozensamplesduringtheearlystagesoftheprotocolandensurethesampleissubmergedduringfreezing.
Substitution: Quicklytransferthesamplesintoglassvialsofmethanolonabedofdryiceinastyrofoambox.Alternatively,thevialscanbeleftinaREVCOfreezer.Sealvialswellandleavefor8-48hours.Smallersampleswillsubstitutefasterthanlargersamples.Increasedcontrastcanbeachievedif1%uranylacetateorosmiumtetroxideisaddedtothesubstitutionmix.Theseheavymetalsdonotinterferewithresinpolymerization.Dryacetonecanbeusedasasubstitutionmediuminsteadofmethanol.However,itisimportantfortheacetonetobedry.
Washing: Washinfreshchangesofsubstitutionmediumpriortoembeddinginresin.Usually2-3changesoverafewhoursissufficient.
Embeddinginresin(usingLowicrylHM20)
Time MeOH:resin
1hr. 1:1
1hr. 1:2
1hr. pureresin
overnight. pureresin
Resinpolymerization: RemovesamplepiecesfromtheglassvialsandplaceinprecooledBEEMcapsulesorsmallEppendorftubescontainingfreshHM20Lowicrylresin.Keepthetubesondryiceandsealthemassoonasthesamplepieceshavebeenadded.Placeonlyonesamplepertube.Arrangethetubesonarackinastyrofoambox,coveredontheinsidewithaluminumfoil,andpackwithdryice.Coverthetubeswithatriangularshapedcardboardboxthathasbeencoveredwithaluminumfoil.PlaceaUVlightabovetheboxandleaveforupto3daystopolymerize.Theresinpolymerizesinindirect360nmUVlight.Toorapidpolymerizationwillresultinshrinkageoftheblockandunevenpolymerization.Oncetheblockshavehardened,polymerizationcancontinueatroomteperaturesusingdirectsunlightortheUVlightusedtosterilizecellculturehoods
Sectioning: Lowicrylresinsdonotcrosslinkwellattissue-resinborders.Itisimportant,whentrimmingtheblock,tomakesurethesampleisnotdislodged.Thebestwaytotrimtheblockisbyusingeitheraglassknifeoroneofthetrimmingdevicescurrentlyavailable(suppliedbyDiatomeUSorHarrisDiamondCo.).Oncetheblockistrimmed,itisfairlyeasytosectionusingroutinesectioningmethods.Diamondknivesarerecommended.TheHM20resinwillnotchangeshapeifwaterissplashedontotheblock.Sectionscanbepickedupfromthewatersurfacewithmetalspecimengrids.ThesectionsarenowreadytobelabeledwithaffinityMarkers.

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Freeze&nbsp;Substitution

Howtopreparesamplesusingfreeze-substitutionwithoutanexpensivemachine.

Freeze Substitution