1. 口 腔 病 理 科 免疫組織化學染色介紹 歡迎出席本課程 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 Immuno Labelling System histochemistry cytochemistry Antibody
(Autolysis)
24 h
Excessive
免疫染色的原理
釣魚作為比喻
Method
Immuno
histochemistry
cytochemistry
Antibody
Antigen
Detection
System
Antibody
Antigen
Polyclonal -
a mix of Ig (rabbit)
Monoclonal -
a single class
of Ig (mouse)
Synthetic\
Commercial
Fixation/Fresh
Antibody
Enzyme (trypsin)
Microwave
Autoclave
Antigen
Antibody 魚餌 魚
Antigen 浮標
Labelling System 釣竿
Method
Labelling System
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 Direct Method Merits Drawback Simple Quick Cheap High
Labelled
Primary
Antibody
Direct Method
Antigen
Cytoplasm
Endothelial cell
Nucleus
Merits
Simple
Quick
Cheap
Drawback
High
background
Human
Labelling
(Only one)
Rabbit/mouse
anti-human
Lower intensity

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

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

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

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

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

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

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

11. Comparison of different methods of IHC
Merits
Drawbacks
Direct method
Simple
Quick
Cheap
High background
Indirect method
PAP
High intensity
Expensive
Time consuming
ABC

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)
Specificity

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

30. Do you have any queries?