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