Immunohistochemistry

Immunohistochemistry is the process of detection of proteins expressed in tissue using antigens/antibodies interaction to visualize localization of expressed proteins. The antibodies, raised to a specific protein antigen can be polyclonal, antibodies raised in non-related animal species, or monoclonal, raised in the species of interest, and then purified. Immunohistochemistry is widely used for diagnosis of cancers. Specific markers are known for various cancers.

• Carcino-embryonic antigen (CEA): used for colon cancer
• CD15 and CD30 : used for Hodgkin’s disease
• Alpha fetoprotein: for yolk sac tumors and hepatocellular carcinoma
• CD117: for gastrointestinal stromal tumors (GIST)

There are basically two methods for immmunohistochemical demonstration of antigens in the tissue, the direct and the indirect method.

The “direct method” is a one-step staining method, and involves a labeled antibody (e.g. FITC conjugated antiserum) reacting directly with the antigen in tissue sections. This technique utilizes only one antibody and the procedure is therefore short and quick. However, it is insensitive due to little signal amplification and rarely used since the introduction of indirect method.

The “indirect method” involves an unlabeled primary antibody (first layer) which reacts with tissue antigen, and a labeled secondary antibody (second layer) reacts with primary antibody. (The secondary antibody must be against the IgG of the animal species in which the primary antibody has been raised.) This method is more sensitive due to signal amplification through several secondary antibody reactions with different antigenic sites on the primary antibody. The second layer antibody can be labeled with a fluorescent dye such as FITC, rhodamine or Texas red, and this is called indirect immunofluorescence method. The second layer antibody may be labeled with an enzyme such as peroxidase, alkaline phosphatase or glucose oxidase, and this is called indirect immunoenzyme method. Offering a substrate to these enzymes will demonstrate presence of the enzyme (and hence the antibody) in a quantifiable fashion.

Immunohistochemical Staining Protocols Immunohistochemistry Image Gallery