Immunochemistry

The process of localizing proteins in cells of a tissue section using the principle of antigens in tissue binding to their respective antibodies is known as Immunohistochemistry (IHC). This technique is widely used for the diagnosis of cancer. The antibodies are tagged with color-producing dyes to make them easily visible. Horseradish peroxidase and phosphatase are the two commonly used color-producing tags. Different fluorophores like FITC are also used to tag the antibodies as an alternate method. This method is widely used in confocal laser scanning microscopy to visualize two interacting protein molecules. Adinocarcinomas, Hodgkin's disease, yolk sac tumors and hepatocellular carcinoma, Gastrointestinal Stromal Tumors (GIST) and prostate cancer are diagnosed by conducting immunohistochemistry using various antigens like Carcinoembryonic antigen, CD 15 & CD30 or Alpha fetoprotein or CD117 or prostate specific antigen accordingly. Direct and indirect methods are the two methods used for the immunohistochemical staining of antigens.

In the direct method, one labelled antibody is used for staining. The antibody directly binds to the antigen which is being stained. Involving only one labeled antibody (FITC conjugated antiserum), this direct method is also known as one-step staining method. Though this method is quick since it uses only one antibody, it is rarely used after the indirect method was introduced.

In the indirect method of immuno-histochemical staining, one antibody is used against the antigen which is being examined and a second labelled antibody is used against the first. The unlabelled primary antibody or the first layer reacts with tissue antigen and the second layer or the labelled secondary antibody reacts with the primary antibody. This indirect method is considered more sensitive, since there is good signal amplification noticed through many secondary antibody reactions with various antigenic sites on the primary antibody.