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1、MethodsinMolecularBiologyMethodsinMolecularBiologyTMVOLUME117ElectronElectronMicroscopyMicroscopyMethodsMethodsandProtocolsandProtocolsEditedbyEditedbyM.A.NasserHajibagheriM.A.NasserHajibagheriHUMANAPRESSPreparationandStainingofSections11GeneralPreparatio
2、nofMaterialandStainingofSectionsHeatherA.Davies1.IntroductionThischapterisaimedatthosewhohavenotpreviouslydoneanyprocessingforelectronmicroscopy(EM).Itdealswithbasicpreparationofmanydifferenttypesofmammalianmaterialforultrastructuralexamination;forprocess
3、ingofplantmaterial(seeHallandHawes,ref.1).Thematerialtobeprocessedmaybecellsuspensions,particulates,monolayercultures,ortissuederivedfromor-gans.Theformerthreemustinitiallybeprocesseddifferentlyfromthelatter.ForEM,theultrastructuremustbepreservedascloseto
4、theinvivosituationaspossible.Thisisdonebyeitherchemicalorcryofixation;thelatterwillbedealtwithinlaterchapters.Aldehydesthatcrosslinkproteinsareusedforchemicalfixation.Glutaraldehyde,adialdehydepreservesultrastucturewellbutpenetratesslowerthanthemonoaldehy
5、de,paraformaldehyde.Glutaralde-hydeisusedaloneforsmallpiecesofmaterial,butamixtureofthetwoalde-hydesmaybeusedforperfusionfixationorfixationoflargeritems.AllreagentsusedforEMprocessingmustbeofhighpurity.Analyticalgradereagentsmustbeusedforallsolutions,e.g.
6、,buffersandstains.GlutaraldehydemustbeEMgrade.Forhigherpurity,distilledorvacuumdistilledqualitiesareavailable.Secondaryfixationisbyosmiumtetroxidewhichreactswithunsat-uratedlipids,iselectron-denseandthusstainsphospholipidsofthecellmem-brane.Thisstepisfoll
7、owedbydehydrationthroughanascendingconcentrationseriesofsolventbeforeembeddinginresin.Forsimplicity,epoxyresin(Epon)embeddingisdescribedinthischapter;otherresinsaredetailedinGlauert(2)andChapters6and7.Ultramicrotomyandstainingultrathinsectionsaredealtwith
8、briefly;foradetailedaccountoftheprocedureandtrouble-shooting(3).Theultrathinsec-From:MethodsinMolecularBiology,vol.117:ElectronMicroscopyMethodsandProtocolsEditedby:N.Hajibagheri©HumanaPressInc.,Totowa,NJ12Daviestio
