An Introduction

1
? ? ? ? ?
An Introduction of Immunocytochemistry

1. ?????????
2. ?????????
3. ?????????

???????
??????????
2

1. ???????

??????
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. ???????
Human
Labelling (Only one)
Rabbit/mouse anti-human
Lower intensity
4
Indirect Method
First labelling
Horse anti-rabbit
Human
Rabbit anti-human
Second labelling
Cytoplasm
Higher intensity
5
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
ABC Method
Antigen
Rabbit anti-human
Horse anti-rabbit
Human
Endothelial cell
Antigen
Cytoplasm
7
Labelling system Peroxidase
Cytoplasm
8
Elimination of Non-Specific Staining
Non-specific binding
Cytoplasm
Specific binding
9
Avidin-Biotin Peroxidase Complex (ABC) method
Biotinylated 2o Ab
Normal serum
H2O2
1o Ab
DAB stain
ABC reagent
Hsu SM, 1981
10
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
Methods Merits Drawbacks
Direct method Simple Quick Cheap High background
Indirect method Simple Quick Cheap High background
PAP High intensity Expensive Time consuming
ABC High intensity Expensive
12
3. ???????
??????
13
3. ???????
??????
14
3. ???????
??????
15
3. ???????
??????
16
3. ???????
??????
17
3. ???????
??????
18
3. ???????
??????
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. ???????
??????
Pleomorphic adenoma of parotid gland (400X)
C (Smooth Muscle Actin, SMA) stain on tumor cells
with myoepithelial differentiation.
A CK (AE1/AE3) stains on tumor cells with ductal
differentiation.
B (S-100 protein) on tumor cells with
myoepithelial differentiation.
20
3. ???????
??????
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. ???????
??????
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. ???????
??????
Ovarian intestinal type mucinous adenocarcinoma
(400X). I (CK7) and J (CK20) coexpress on tumor
with heterogenous cytoplasmic staining pattern.
23
3. ???????
??????
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. ???????
??????
Nodular sclerosing type of classical Hodgkins
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. ???????
??????
26
(No Transcript)
27
(No Transcript)
28
(No Transcript)
29
-
30
(No Transcript)