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ICCImmunocytochemistry

ICCImmunocytochemistry
is a common laboratory technique that is used to anatomically localize presence of a specific protein or antigen in cells by use of a specific primary antibody that binds to it. The primary antibody allows visualization of the protein under a fluorescence microscope when it is bound by a secondary antibody that has a conjugated fluorophore. ICC allows researchers to evaluate whether or not cells in a particular sample express the antigen in question. In cases where an immunopositive signal is found, ICC also allows researchers to determine which sub-cellular compartments are expressing the antigen.

Immunocytochemistry (ICC): a highly sensitive and specific method to localize the antigen in the cells with a labeled antibody detect cellular antigen (cell surface or intracellular) also it is easy and rapid.

Principle

The primary (monoclonal antibody) binds the antigen The secondary antibody (conjugated or labeled by enzyme for example) binds to the bound primary antibody The chromogenic substrate of the enzyme when reacts with the bound enzyme (the lablel) will yield a colored precipitate at the site of the antigen. So its name (ICC) !!


mAb
2nd Ab
conjugate
6
Substrate Chromogen
Principle of ICC
Ag

CD3 positive PBL

IHC Immunohistochemistry

IHC


Immunohistochemistry (IHC): a highly sensitive and specific method to localize the antigen in the tissue sections with a labeled antibody (to detect the virus antigen) So its name (IHC) !!

Major components in a complete immunohistochemistry experiment:

1)Primary antibody binds to specific antigen; 2)The antibody-antigen complex is formed by incubation with a secondary, enzyme-conjugated, antibody; 3)With presence of substrate and chromogen, the enzyme catalyzes to generate colored deposits at the sites of antibody-antigen binding.

mAb
2nd Ab
4
6
5
Principle of IHC
Ag



1

2
Xylene
99%
95%
70%
DW
Tissue
3
Monoclonal Ab
1 Hour

Procedure of IHC

Tissue
PBS tween
5
Conjugate
6
Substrate
7
Counter stain
4
2nd Ab



Immunohistochemical staining of Placental tissue taken from a woman had spontaneous abortion using monoclonal antibody to HCMV early protein, showing positive trophoblasts (pictures 1 & 2) and tissue infiltrating neutrophils (picture 3).

Applications

Detection of the virus in different tissues indicating a role of these viruses in the pathogenesis of certain diseases in these tissues, examples: 1.Detection of EBV in patients with lymphoma and nasopharyngeal carcinoma. 2.Detection of HCMV and rubella virus in the placental tissue of aborted or stillbirth babies. 3.Detection of HCMV in renal transplanted patients who had acute HCMV.

ISH(In situ Hybridization)

In situ Hybridization (ISH): a highly sensitive and specific method detects, as target nucleic acid, the viral DNA or RNA in tissue sections or cytological preparations. It is useful in detecting the viruses that are difficult to grow. Examples: detecting HCMV DNA in urine and puffy-coat specimens from neonates and bone marrow transplant recipients. Detection of different types of HPV in cancer of the cervix.

5
1
Target DNA or mRNA
2
Biotin- Probe
3
Conjugate
4
Substrate
Principle of ISH


In Situ Hybridization Procedure


1

2
Xylen
99%
95%
70%
DW
Tissue
3
Proteinase K


4
Hybridization
Cover slip
5
6


7

2
Conjugate
3
Substrate
4
Counter stain
5
Detergent wash
1