口 腔 病 理 科

免疫組織化學染色介紹

An Introductionof

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

Immunohistochemistrycytochemistry

AntibodyAntigen

AntibodyAntigenDetectionSystem

Antigen

Antibody

釣魚作為比喻

Antibody魚餌魚 Antigen

浮標 Labelling System

釣竿 Method

Method

Fixation/FreshPolyclonal - a mix of Ig (rabbit)

Monoclonal - a single classof Ig (mouse)

Synthetic\ Commercial

Enzyme: Horseradish Peroxidase (HP), Alkaline Phosphatase (AP);FITC, TRITC (tetramethyl rhadamineisothiocyanate); gold; isotope

Chromagen: Diamino benzidine(DAB)

(Autolysis)

24 h

Excessive

Enzyme (trypsin)MicrowaveAutoclave

1.免疫染色的原理

Merits Simple Quick Cheap

Drawback High background

Lower intensity

Direct Method

An

tigen

Cytoplasm

An

tigen

En

do

thelial cell

Nucleus

Labelling(Only one)

LabelledPrimary Antibody

Human

Rabbit/mouseanti-human

2. 免疫染色的種類

Higher intensity

Merits Simple Quick Cheap

Drawback High background

Indirect Method

Cytoplasm

An

tigen

An

tigen

En

do

thelial cell

Nucleus

Unlabelled Primary Antibody

First labelling

Second labelling

Human

Rabbitanti-human

Horseanti-rabbit

Lab

elled S

econ

dary

An

tibo

dy

PAP Method

An

tigen

Cytoplasm

An

tigen

En

do

thelial cell

Nucleus

UnlabelledSecondary (Bridging) Antibody

PeroxidaseAnti-Peroxidase(PAP)

UnlabelledPrimary AntibodyFirst labelling

Second labelling

Third labelling

Higher intensity

Drawback Expensive Time consuming

Human

Rabbitanti-human

Horseanti-rabbit

RabbitPAP

ABC Method

An

tigen

Cytoplasm

An

tigen

En

do

thelial cell

Nucleus

UnlabelledPrimary Antibody

Higher intensity

Drawback Expensive

Human

Rabbitanti-human Horse

anti-rabbit

BiotinylatedSecondary(Bridging) Antibody

Avidin-BiotinylatedPeroxidaseComplex (ABC)

Cytoplasm

Nucleus

Antigen AntigenEndothelial cell

Elimination of Endogenous Peroxidase

Lab

elle

dP

rim

ary

An

tib

od

y

Endogenousperoxidase

H2O2

H2O2

H2O2

H2O2

Eliminate endogeousperoxidase

Labelling system:Peroxidase

Drawback High background

Cytoplasm

Nucleus

Elimination of Non-Specific Staining

Antigen AntigenEndothelial cell Antigen AntigenEndothelial cell

Lab

elle

dP

rim

ary

An

tib

od

yL

abel

led

Pri

mar

y A

nti

bo

dy

Normal Serum (same species of 2nd Ab)

Endothelial cellAntigen Collagen Endothelial cell

Lab

elle

dP

rim

ary

An

tib

od

y

Lab

elle

dP

rim

ary

An

tib

od

y

Specific bindingNon-specific binding

Avidin-Biotin Peroxidase Complex (ABC) method

H2O2Normal serum

1o Ab Biotinylated 2o Ab

ABCreagent

DABstain

Hsu SM, 1981

PAP Method

An

tigen

An

tigen

Lym

ph

oid

cell

Nucleus

UnlabelledPrimary Antibody

Human

Rabbitanti-human

Horseanti-rabbit

Rabbit APAAP

UnlabelledSecondary (Bridging)Antibody (1)

Cytoplasm

UnlabelledSecondary (BridgingAntibody (2)

Rabbit PAP

APAAP

PAP

Alkaline Phosphaste AntiAlkaline Phosphaste

Comparison of different methods of IHC

Methods Merits Drawbacks

Direct method SimpleQuick Cheap

High background

Indirect method SimpleQuick Cheap

High background

PAP High intensity

ExpensiveTime consuming

ABC High intensity

Expensive

3. 免疫染色的應用 了解細胞來源

3. 免疫染色的應用 了解細胞來源

3. 免疫染色的應用 了解細胞來源

3. 免疫染色的應用 了解細胞來源

3. 免疫染色的應用 了解細胞來源

3. 免疫染色的應用 了解細胞來源

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 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 isnegative 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 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 myoepithelialdifferentiation.

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) andF (synaptophysin) show granularcytoplasmic 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 differentiatedsquamous cell carcinoma (400X).p16 shows strong full thickness overexpression on the cytoplasm andnucleus 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 heterogenouscytoplasmic staining pattern.

3. 免疫染色的應用 了解細胞來源K: Colonic adenocarcinoma (400X).CK20 shows diffuse and strongcytoplasmic 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 paranuclearspace 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.

3. 免疫染色的應用

了解細胞來源

Table 1 Antibodies used, sources and their specificity

Antibodies Sources (dilution) Specificity

Conclusion

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Table 1 Antibodies used, their specificity and resultsAntibodies Sources (dilution) Specificity Results