口腔病理科免疫組織化學染色介紹AnIntroductionofImmunocytochemistry歡迎出席本課程明白免疫染色的原理瞭解免疫染色的種類知道免疫染色的應用ImmunohistochemistrycytochemistryAntibodyAntigenAntibodyAntigenDetectionSystemAntigenAntibody釣魚作為比喻Antibody魚餌魚Antigen浮標LabellingSystem釣竿MethodMethodFixation/FreshLabellingSystemPolyclonal-amixofIg(rabbit)Monoclonal-asingleclassofIg(mouse)mercialEnzyme:HorseradishPeroxidase(HP),AlkalinePhosphatase(AP);FITC,TRITC(tetramethylrhadamineisothiocyanate);gold;isotopeChromagen:Diaminobenzidine(DAB)(Autolysis)24hExcessiveEnzyme(trypsin)MicrowaveAutoclave免疫染色的原理MeritsSimpleQuickCheapDrawbackHighbackgroundLowerintensityDirectMethodAntigenCytoplasmAntigenEndothelialcellNucleusLabelling(Onlyone)LabelledPrimaryAntibodyHumanRabbit/mouseanti--humanHorseanti-rabbitLabelledSecondaryAntibodyPAPMethodAntigenCytoplasmAntigenEndothelialcellNucleusUnlabelledSecondary(Bridging)AntibodyPeroxidaseAnti-Peroxidase(PAP)UnlabelledPrimaryAntibodyFirstlabellingSecondlabellingThirdlabellingHigherintensityDrawbackExpensiveTimeconsumingHumanRabbitanti-humanHorseanti-rabbitRabbitPAPABCMethodAntigenCytoplasmAntigenEndothelialcellNucleusUnlabelledPrimaryAntibodyHigherintensityDrawbackExpensiveHumanRabbitanti-humanHorseanti-rabbitBiotinylatedSecondary(Bridging)AntibodyAvidin-plex(ABC)CytoplasmNucleusAntigenAntigenEndothelialcellEliminationofEndogenousPeroxidaseLabelledPrimaryAntibodyEndogenousperoxidaseH2O2H2O2H2O2H2O2EliminateendogeousperoxidaseLabellingsystem:PeroxidaseDrawbackHighbackgroundCytoplasmNucleusEliminationofNon-SpecificStainingAntigenAntigenEndothelialcellAntigenAntigenEndothelialcellLabelledPrimaryAntibodyLabelledPrimaryAntibodyNormalSerum(samespeciesof2ndAb)EndothelialcellAntigenCollagenEndothelialcellLabelledPrimaryAntibodyLabelledPrimaryAntibodySpecificbindingNon-specificbindingAvidin-plex(ABC)methodH2O2Norm
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