1、口口 腔腔 病病 理理 科科免免疫疫組組織織化化學學染染色色介紹介紹An IntroductionofImmunocytochemistry歡迎出席本課程歡迎出席本課程1.明白免疫染色的原理明白免疫染色的原理2.瞭解免疫染色的種類瞭解免疫染色的種類3.知道免疫染色的應用知道免疫染色的應用ImmunohistochemistrycytochemistryAntibodyAntigenAntibodyAntigenDetectionSystemAntigenAntibody釣魚作為比喻釣魚作為比喻Antibody魚餌魚餌魚魚Antigen浮標浮標Labelling System釣竿釣竿Method
2、MethodFixation/FreshPolyclonal-a mix of Ig(rabbit)Monoclonal-a single classof Ig(mouse)Synthetic CommercialEnzyme:Horseradish Peroxidase(HP),Alkaline Phosphatase(AP);FITC,TRITC(tetramethyl rhadamineisothiocyanate);gold;isotope Chromagen:Diamino benzidine(DAB)(Autolysis)24 hExcessiveuEnzyme(trypsin)u
3、MicrowaveuAutoclave1.免疫染色的原理免疫染色的原理Merits Simple Quick CheapDrawback High backgroundLower intensityDirect MethodAntigenCytoplasmAntigenEndothelial cellNucleusLabelling(Only one)LabelledPrimary AntibodyHumanRabbit/mouseanti-human2.免疫染色的種類免疫染色的種類Higher intensityMerits Simple Quick CheapDrawback High b
4、ackgroundIndirect MethodCytoplasmAntigenAntigenEndothelial cellNucleusUnlabelled Primary AntibodyFirst labellingSecond labellingHumanRabbitanti-humanHorseanti-rabbitLabelled SecondaryAntibodyPAP MethodAntigenCytoplasmAntigenEndothelial cellNucleusUnlabelledSecondary(Bridging)AntibodyPeroxidaseAnti-P
5、eroxidase(PAP)UnlabelledPrimary AntibodyFirst labellingSecond labellingThird labellingHigher intensityDrawback Expensive Time consumingHumanRabbitanti-humanHorseanti-rabbitRabbitPAPABC MethodAntigenCytoplasmAntigenEndothelial cellNucleusUnlabelledPrimary AntibodyHigher intensityDrawback ExpensiveHum
6、anRabbitanti-humanHorseanti-rabbitBiotinylatedSecondary(Bridging)AntibodyAvidin-BiotinylatedPeroxidaseComplex(ABC)CytoplasmNucleusAntigenAntigenEndothelial cellElimination of Endogenous PeroxidaseLabelledPrimary AntibodyEndogenousperoxidaseH2O2H2O2H2O2H2O2Eliminate endogeousperoxidase Labelling syst
7、em:PeroxidaseDrawback High backgroundCytoplasmNucleusElimination of Non-Specific StainingAntigenAntigenEndothelial cellAntigenAntigenEndothelial cellLabelledPrimary AntibodyLabelledPrimary AntibodyNormal Serum(same species of 2nd Ab)Endothelial cellAntigen Collagen Endothelial cellLabelledPrimary An
8、tibodyLabelledPrimary AntibodySpecific bindingNon-specific bindingAvidin-Biotin Peroxidase Complex(ABC)methodH2O2Normal serum1o AbBiotinylated 2o AbABCreagentDABstainHsu SM,1981PAP MethodAntigenAntigenLymphoid cellNucleusUnlabelledPrimary AntibodyHumanRabbitanti-humanHorseanti-rabbitRabbit APAAPUnla
9、belledSecondary(Bridging)Antibody(1)CytoplasmUnlabelledSecondary(BridgingAntibody(2)Rabbit PAPAPAAPPAPAlkaline Phosphaste AntiAlkaline PhosphasteComparison of different methods of IHCMethodsMeritsDrawbacksDirect methodSimpleQuick CheapHigh backgroundIndirect methodSimpleQuick CheapHigh backgroundPAP
10、High intensityExpensiveTime consumingABCHigh intensityExpensive3.免疫染色的應用免疫染色的應用了解細胞來源了解細胞來源3.免疫染色的應用免疫染色的應用了解細胞來源了解細胞來源3.免疫染色的應用免疫染色的應用了解細胞來源了解細胞來源3.免疫染色的應用免疫染色的應用了解細胞來源了解細胞來源3.免疫染色的應用免疫染色的應用了解細胞來源了解細胞來源3.免疫染色的應用免疫染色的應用了解細胞來源了解細胞來源3.免疫染色的應用免疫染色的應用了解細胞來源了解細胞來源A:CK(AE1/AE3)shows diffuse cytoplasmic st
11、aining pattern at full thickness of the squamous mucosa.B:p63 shows nuclear staining pattern at basalto intermediate layer of squamous mucosa.C:EMA shows membranous staining pattern of epithelial cells.D:Vimentin shows diffuse cytoplasmic staining pattern on subepithelialstromal cells.The epithelium
12、 isnegative for vimentinOral mucosa(100X)3.免疫染色的應用免疫染色的應用了解細胞來源了解細胞來源C(Smooth Muscle Actin,SMA)stain on tumor cells with myoepithelialdifferentiation.Pleomorphic adenoma of parotid gland(400X)A:CK(AE1/AE3)stains on tumor cells with ductal differentiation.B(S-100 protein)on tumor cells with myoepithe
13、lialdifferentiation.3.免疫染色的應用免疫染色的應用了解細胞來源了解細胞來源D:Vimentin shows cytoplasmic staining pattern on sarcomatoid carcinoma.Neuroendocrine carcinoma of breast(400X).E(chromogranin)andF(synaptophysin)show granularcytoplasmic staining pattern on tumor cells.3.免疫染色的應用免疫染色的應用了解細胞來源了解細胞來源Metastatic ovarian co
14、lonic type mucinous carcinoma(400X).Carcinoembronic antigen(CEA)stains on apical surface of adenocarcinoma.Esophageal moderately differentiatedsquamous cell carcinoma(400X).p16 shows strong full thickness overexpression on the cytoplasm andnucleus of tumor cells.The adjacent mucous gland is negative
15、 for p16.3.免疫染色的應用免疫染色的應用了解細胞來源了解細胞來源Ovarian intestinal type mucinous adenocarcinoma(400X).I(CK7)and J(CK20)coexpress on tumor with heterogenouscytoplasmic staining pattern.3.免疫染色的應用免疫染色的應用了解細胞來源了解細胞來源K:Colonic adenocarcinoma(400X).CK20 shows diffuse and strongcytoplasmic staining pattern of tumor c
16、ells.L:Malignant melanoma(400X).HMB-45 shows cytoplasmic staining pattern on melanoma cells.3.免疫染色的應用免疫染色的應用了解細胞來源了解細胞來源N(CD15)and O(CD30)coexpress on cell membrane and paranuclearspace of RS cellsNodular sclerosing type of classical Hodgkins lymphomaM:LCA stains on all lymphoid cells including large malignant RS cells and surround reactive small lymphocytes.3.免疫染色的應用免疫染色的應用了解細胞來源了解細胞來源Table 1 Antibodies used,sources and their specificityAntibodiesSources(dilution)SpecificityConclusion-Table 1 Antibodies used,their specificity and resultsAntibodiesSources(dilution)SpecificityResults
