ICC Immunocytochemistry

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.

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) !!

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

CD3 positive PBL

Immunohistochemistry IHC Immunohistochemistry

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.  

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.

Principle of IHC 6 5 4 2nd Ab mAb Ag

Oven 1 Hour Procedure of IHC 65ºC Monoclonal Ab Incubator 37ºC Xylene 2 Monoclonal Ab Xylene Oven 65ºC 99% Incubator 37ºC 95% 70% 1 Hour Tissue DW

Conjugate Substrate Counter stain 2nd Ab 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.

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

In Situ Hybridization Procedure

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

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

Conjugate Substrate Counter stain 1 5 2 Conjugate 3 Substrate Detergent wash 4 Counter stain