Freeze Substitution
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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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