口 腔 病 理 科

1. 口腔病理科 An Introduction of Immunocytochemistry • 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 歡迎出席本課程 免疫組織化學染色介紹

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

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

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

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

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

7. Labelled Primary Antibody Elimination of Endogenous Peroxidase Nucleus Antigen Drawback High background H2O2 H2O2 Endothelial cell Eliminate endogeous peroxidase H2O2 Endogenous peroxidase H2O2 Antigen Labelling system: Peroxidase Cytoplasm

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

9. Avidin-BiotinPeroxidaseComplex(ABC)method Biotinylated 2o Ab Normal serum H2O2 1o Ab DAB stain ABC reagent Hsu SM, 1981

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

11. Comparison of different methods of IHC

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 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. C (Smooth Muscle Actin, SMA) stain 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 Nodular sclerosing type of classical Hodgkin’s lymphoma N (CD15) and O (CD30) coexpress on cell membrane and paranuclear space of RS cells 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

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

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

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