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Immunohistochemistry refers to the process of localizing proteins in cells of a tissue section exploiting the principle of antigens in tissue binding to their respective antibodies. Visualization is enabled by tagging the antibody with color producing tags. Typical examples include Horseradish peroxidase or alklaline phosphatase. An ideal chemistry produces the required color using different redox dyes. Alternatively, the antibody can also be tagged to different fluorophores, e.g. FITC. This method is of great use in confocal laser scanning microscopy which is more sensitive and helps visualize two interacting protein molecules together.

The antibodies can be polyclonal or monoclonal in origin, the monoclonal ones being more specific in nature. Immunohistochemistry is widely used for diagnosis of cancers. Specific markers are known for various cancers. However, the technique is also widely used in basic research to understand the distribution and localization of biomarkers in different parts of a tissue.

Methods

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

The "direct method" is a one-step staining method, and involves a labeled antibody (i.e. 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 the indirect method.


The "indirect method" involves an unlabeled primary antibody (first layer) which reacts with tissue antigen, and a labeled secondary antibody (second layer) which reacts with the 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 the 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 the indirect immunoenzyme method. Offering a substrate to these enzymes will demonstrate presence of the enzyme (and hence the antibody) in a quantifiable fashion.

External links

Anatomical pathology | Staining | Immunohistochimie | Antikörperfärbung

 

This article is licensed under the GNU Free Documentation License. It uses material from the "Immunohistochemistry".

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