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《MMB vol.115 Javois L.C. (eds.) Immunocytochemical Methods and Protocols (Humana Press, 1999)(ISBN 0896038130)(447s)》由会员上传分享，免费在线阅读，更多相关内容在学术论文-天天文库。
1、MethodsinMolecularBiologyMethodsinMolecularBiologyTMVOLUME115ImmunocytochemicalImmunocytochemicalMethodsandMethodsandProtocolsProtocolsSecondEditionSecondEditionEditedbyEditedbyLoretteC.JavoisLoretteC.JavoisHUMANAPRESSHUMANAPRESSAntibodies31OverviewofAntibodyU
2、seinImmunocytochemistrySu-YauMao,LoretteC.Javois,andUteM.Kent1.IntroductionImmunocytochemistry,bydefinition,istheidentificationofatissuecon-stituentinsitubymeansofaspecificantigen–antibodyinteractionwheretheantibodyhasbeentaggedwithavisiblelabel(1).Cellstainin
3、gisapowerfulmethodtodemonstrateboththepresenceandsubcellularlocationofaparticu-larmoleculeofinterest(2).Initialattemptstolabelantibodieswithordinarydyeswereunsatisfactorybecausethelabelwasnotsufficientlyvisibleunderthemicroscope.A.H.Coonsfirstintroducedimmunof
4、luorescencein1941,usingspecificantibodieslabeledwithafluorescentdyetolocalizesubstancesintissues(3).Thistechniquewasconsidereddifficult,anditspotentialwasnotwidelyrealizedfornearly20yr.Earlyattemptsfocusedonlabelingthespe-cificantibodyitselfwithafluorophore(se
5、eChapter6).Thelabeledantibodywasthenappliedtothetissuesectiontoidentifytheantigenicsites(directmethod)(3)(seeChapter15).Later,themoresensitiveandversatileindirectmethodwasintroduced(4)(seeChapters16–18).Inthismethod,thespecificantibody,boundtotheantigen,wasdet
6、ectedwithasecondaryreagent,usuallyanotherantibodythathadbeentaggedwitheitherafluorophoreoranenzyme.Fluorochrome-labeledanti-immunoglobulinantibodiesarenowwidelyusedinimmunocytochemistry,flowcytometry(seeChapters30–39),andhybri-domascreening.Theavailabilityoffl
7、uorophoreswithdifferentemissionspec-trahasalsomadeitpossibletodetecttwoormoreantigensonthesamecellortissuesection(seeChapter14).Althoughfluorescentlabelingofferssensitiv-ityandhighresolution,thereareseveraldisadvantages.First,itrequiresspe-cialinstrumentation:
8、afluorescencemicroscope,aconfocalmicroscope,oraflowcytometer.Second,backgrounddetailsaredifficulttoappreciate,andcel-lularautofluorescencecansometimesmaketheinterpretationdifficult
