1. ICC Immunocytochemistry

2. ICC Immunocytochemistry
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.

3. 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.

4. 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) !!

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

6. CD3 positive PBL

7. Immunohistochemistry
IHC
Immunohistochemistry

8. IHC
Immunohistochemistry (IHC) is a wide-used biological technique that combines anatomy, physiology, immunology and biochemistry. Developed from the antigen-antibody binding reaction, immunohistochemistry can be considered as a method that visualize distribution and localization of specific antigen or cellular components in separated tissues, or tissue sections. Compared to other bio-techniques that are based on the antigen-antibody reaction such as immune precipitation, or western-blot.

9. 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) !!

10. 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.

11. Principle of IHC 6 5 4
2nd Ab
mAb Ag

12. Oven 1 Hour Procedure of IHC 65ºC Monoclonal Ab Incubator 37ºC Xylene2
Monoclonal Ab
Xylene
Oven
65ºC
99%
Incubator
37ºC
95%
70%
1 Hour
Tissue DW

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

14. 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).

16. 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.

17. ISH (In situ Hybridization)

18. 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.

19. Principle of ISH Substrate Conjugate Biotin- Probe Target DNA or mRNA4 5
Substrate
Conjugate 3
Biotin-
Probe 2
Target
DNA or
mRNA 1

20. In Situ Hybridization Procedure

21. 3 1 2
Xylen
Proteinase K
Oven
65ºC
99%
95%
70%
Tissue DW

22. 6 4 5
Hybridization
Cover slip
Oven
70ºC 7
Incubator
37ºC

23. Conjugate Substrate Counter stain1 5 2
Conjugate 3
Substrate
Detergent
wash 4
Counter stain