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口 腔 病 理 科口 腔 病 理 科 免疫組織化學染色介紹 An Introduction of Immunocytochemistry 歡迎出席本課程 1. 明白免疫染色的原理 2. 瞭解免疫染色的種類 3. 知道免疫染色的應用.

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Presentation on theme: "口 腔 病 理 科口 腔 病 理 科 免疫組織化學染色介紹 An Introduction of Immunocytochemistry 歡迎出席本課程 1. 明白免疫染色的原理 2. 瞭解免疫染色的種類 3. 知道免疫染色的應用."— Presentation transcript:

1 口 腔 病 理 科口 腔 病 理 科 免疫組織化學染色介紹 An Introduction of Immunocytochemistry 歡迎出席本課程 1. 明白免疫染色的原理 2. 瞭解免疫染色的種類 3. 知道免疫染色的應用

2 Immuno histochemistry cytochemistry AntibodyAntigen Antibody Antigen Detection System Antigen Antibody 釣魚作為比喻 Antibody 魚餌 魚 Antigen 浮標 Labelling System 釣竿 Method Fixation/Fresh Polyclonal - a mix of Ig (rabbit) Monoclonal - a single class of Ig (mouse) Synthetic\ Commercial Enzyme: Horseradish Peroxidase (HP), Alkaline Phosphatase (AP);FITC, TRITC (tetramethyl rhadamine isothiocyanate); gold; isotope Chromagen: Diamino benzidine (DAB) (Autolysis) 24 h Excessive  Enzyme (trypsin)  Microwave  Autoclave 1. 免疫染色的原理

3 Merits Simple Quick Cheap Drawback High background Lower intensity Direct Method Antigen Cytoplasm Antigen Endothelial cell Nucleus Labelling (Only one) Labelled Primary Antibody Human Rabbit/mouse anti-human 2. 免疫染色的種類

4 Higher intensity Merits Simple Quick Cheap Drawback High background Indirect Method Cytoplasm Antigen Endothelial cell Nucleus Unlabelled Primary Antibody First labelling Second labelling Human Rabbit anti-human Horse anti-rabbit Labelled Secondary Antibody

5 PAP Method Antigen Cytoplasm Antigen Endothelial cell Nucleus Unlabelled Secondary (Bridging) Antibody Peroxidase Anti-Peroxidase (PAP) Unlabelled Primary Antibody First labelling Second labelling Third labelling Higher intensity Drawback Expensive Time consuming Human Rabbit anti-human Horse anti-rabbit Rabbit PAP

6 ABC Method Antigen Cytoplasm Antigen Endothelial cell Nucleus Unlabelled Primary Antibody Higher intensity Drawback Expensive Human Rabbit anti-human Horse anti-rabbit Biotinylated Secondary (Bridging) Antibody Avidin- Biotinylated Peroxidase Complex (ABC)

7 Cytoplasm Nucleus Antigen Endothelial cell Elimination of Endogenous Peroxidase Labelled Primary Antibody Endogenous peroxidase H2O2H2O2 H2O2H2O2 H2O2H2O2 H2O2H2O2 Eliminate endogeous peroxidase Labelling system: Peroxidase Drawback High background

8 Cytoplasm Nucleus Elimination of Non-Specific Staining Antigen Endothelial cell Antigen Endothelial cell Labelled Primary Antibody Labelled Primary Antibody Normal Serum (same species of 2nd Ab) Endothelial cell Antigen Collagen Endothelial cell Labelled Primary Antibody Labelled Primary Antibody Specific binding Non-specific binding

9 Avidin-Biotin Peroxidase Complex (ABC) method H2O2H2O2 Normal serum 1 o Ab Biotinylated 2 o Ab ABC reagent DAB stain Hsu SM, 1981

10 PAP Method Antigen Lymphoid cell Nucleus Unlabelled Primary Antibody Human Rabbit anti-human Horse anti-rabbit Rabbit APAAP Unlabelled Secondary (Bridging) Antibody (1) Cytoplasm Unlabelled Secondary (Bridging Antibody (2) Rabbit PAP APAAP PAP Alkaline Phosphaste Anti Alkaline Phosphaste

11 Comparison of different methods of IHC MethodsMeritsDrawbacks Direct methodSimple Quick Cheap High background Indirect methodSimple Quick Cheap High background PAPHigh intensity Expensive Time consuming ABCHigh intensity Expensive

12 3. 免疫染色的應用了解細胞來源

13 3. 免疫染色的應用了解細胞來源

14 3. 免疫染色的應用了解細胞來源

15 3. 免疫染色的應用了解細胞來源

16 3. 免疫染色的應用了解細胞來源

17 3. 免疫染色的應用了解細胞來源

18 3. 免疫染色的應用了解細胞來源 A: CK (AE1/AE3) shows diffuse cytoplasmic staining pattern at full thickness of the squamous mucosa. B: p63 shows nuclear staining pattern at basal to intermediate layer of squamous mucosa. C: EMA shows membranous staining pattern of epithelial cells. D: Vimentin shows diffuse cytoplasmic staining pattern on subepithelial stromal cells. The epithelium is negative for vimentin Oral mucosa (100X)

19 3. 免疫染色的應用了解細胞來源 C (Smooth Muscle Actin, SMA) stain on tumor cells with myoepithelial differentiation. 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 myoepithelial differentiation.

20 3. 免疫染色的應用了解細胞來源 D: Vimentin shows cytoplasmic staining pattern on sarcomatoid carcinoma. Neuroendocrine carcinoma of breast (400X). E (chromogranin) and F (synaptophysin) show granular cytoplasmic staining pattern on tumor cells.

21 3. 免疫染色的應用了解細胞來源 Metastatic ovarian colonic type mucinous carcinoma (400X). Carcinoembronic antigen (CEA) stains on apical surface of adenocarcinoma. Esophageal moderately differentiated squamous cell carcinoma (400X). p16 shows strong full thickness overexpression on the cytoplasm and nucleus of tumor cells. The adjacent mucous gland is negative for p16.

22 3. 免疫染色的應用了解細胞來源 Ovarian intestinal type mucinous adenocarcinoma (400X). I (CK7) and J (CK20) coexpress on tumor with heterogenous cytoplasmic staining pattern.

23 3. 免疫染色的應用了解細胞來源 K: Colonic adenocarcinoma (400X). CK20 shows diffuse and strong cytoplasmic staining pattern of tumor cells. L: Malignant melanoma (400X). HMB-45 shows cytoplasmic staining pattern on melanoma cells.

24 3. 免疫染色的應用了解細胞來源 N (CD15) and O (CD30) coexpress on cell membrane and paranuclear space of RS cells Nodular sclerosing type of classical Hodgkin’s lymphoma M: LCA stains on all lymphoid cells including large malignant RS cells and surround reactive small lymphocytes.

25 3. 免疫染色的應用 了解細胞來源

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27 Table 1 Antibodies used, sources and their specificity Antibodies Sources (dilution) Specificity

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29 Conclusion - Table 1 Antibodies used, their specificity and results Antibodies Sources (dilution) Specificity Results

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