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1、MethodsinMolecularBiologyMethodsinMolecularBiologyTMVOLUME117ElectronElectronMicroscopyMicroscopyMethodsMethodsandProtocolsandProtocolsEditedbyEditedbyM.A.NasserHajibagheriM.A.NasserHajibagheriHUMANAPRESSPreparationandStainingofSections11GeneralPreparationofMaterialandStainingof
2、SectionsHeatherA.Davies1.IntroductionThischapterisaimedatthosewhohavenotpreviouslydoneanyprocessingforelectronmicroscopy(EM).Itdealswithbasicpreparationofmanydifferenttypesofmammalianmaterialforultrastructuralexamination;forprocessingofplantmaterial(seeHallandHawes,ref.1).Themat
3、erialtobeprocessedmaybecellsuspensions,particulates,monolayercultures,ortissuederivedfromor-gans.Theformerthreemustinitiallybeprocesseddifferentlyfromthelatter.ForEM,theultrastructuremustbepreservedasclosetotheinvivosituationaspossible.Thisisdonebyeitherchemicalorcryofixation;th
4、elatterwillbedealtwithinlaterchapters.Aldehydesthatcrosslinkproteinsareusedforchemicalfixation.Glutaraldehyde,adialdehydepreservesultrastucturewellbutpenetratesslowerthanthemonoaldehyde,paraformaldehyde.Glutaralde-hydeisusedaloneforsmallpiecesofmaterial,butamixtureofthetwoalde-h
5、ydesmaybeusedforperfusionfixationorfixationoflargeritems.AllreagentsusedforEMprocessingmustbeofhighpurity.Analyticalgradereagentsmustbeusedforallsolutions,e.g.,buffersandstains.GlutaraldehydemustbeEMgrade.Forhigherpurity,distilledorvacuumdistilledqualitiesareavailable.Secondaryf
6、ixationisbyosmiumtetroxidewhichreactswithunsat-uratedlipids,iselectron-denseandthusstainsphospholipidsofthecellmem-brane.Thisstepisfollowedbydehydrationthroughanascendingconcentrationseriesofsolventbeforeembeddinginresin.Forsimplicity,epoxyresin(Epon)embeddingisdescribedinthisch
7、apter;otherresinsaredetailedinGlauert(2)andChapters6and7.Ultramicrotomyandstainingultrathinsectionsaredealtwithbriefly;foradetailedaccountoftheprocedureandtrouble-shooting(3).Theultrathinsec-From:MethodsinMolecularBiology,vol.117:ElectronMicroscopyMethodsandProtocolsEditedby:N.H
8、ajibagheri©HumanaPressInc.,Totowa,NJ12Daviestio