口 腔 病 理 科口 腔 病 理 科 免疫組織化學染色介紹 an introduction of...
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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