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1、MethodsinMolecularBiologyMethodsinMolecularBiologyTMVOLUME115ImmunocytochemicalImmunocytochemicalMethodsandMethodsandProtocolsProtocolsSecondEditionSecondEditionEditedbyEditedbyLoretteC.JavoisLoretteC.JavoisHUMANAPRESSHUMANAPRESSAntibodies31Overviewof
2、AntibodyUseinImmunocytochemistrySu-YauMao,LoretteC.Javois,andUteM.Kent1.IntroductionImmunocytochemistry,bydefinition,istheidentificationofatissuecon-stituentinsitubymeansofaspecificantigen–antibodyinteractionwheretheantibodyhasbeentaggedwithavisiblela
3、bel(1).Cellstainingisapowerfulmethodtodemonstrateboththepresenceandsubcellularlocationofaparticu-larmoleculeofinterest(2).Initialattemptstolabelantibodieswithordinarydyeswereunsatisfactorybecausethelabelwasnotsufficientlyvisibleunderthemicroscope.A.H.
4、Coonsfirstintroducedimmunofluorescencein1941,usingspecificantibodieslabeledwithafluorescentdyetolocalizesubstancesintissues(3).Thistechniquewasconsidereddifficult,anditspotentialwasnotwidelyrealizedfornearly20yr.Earlyattemptsfocusedonlabelingthespe-ci
5、ficantibodyitselfwithafluorophore(seeChapter6).Thelabeledantibodywasthenappliedtothetissuesectiontoidentifytheantigenicsites(directmethod)(3)(seeChapter15).Later,themoresensitiveandversatileindirectmethodwasintroduced(4)(seeChapters16–18).Inthismethod
6、,thespecificantibody,boundtotheantigen,wasdetectedwithasecondaryreagent,usuallyanotherantibodythathadbeentaggedwitheitherafluorophoreoranenzyme.Fluorochrome-labeledanti-immunoglobulinantibodiesarenowwidelyusedinimmunocytochemistry,flowcytometry(seeCha
7、pters30–39),andhybri-domascreening.Theavailabilityoffluorophoreswithdifferentemissionspec-trahasalsomadeitpossibletodetecttwoormoreantigensonthesamecellortissuesection(seeChapter14).Althoughfluorescentlabelingofferssensitiv-ityandhighresolution,therea
8、reseveraldisadvantages.First,itrequiresspe-cialinstrumentation:afluorescencemicroscope,aconfocalmicroscope,oraflowcytometer.Second,backgrounddetailsaredifficulttoappreciate,andcel-lularautofluorescencecansometimesmaketheinterpretationdifficult
