Explaining Immunostaining Protocols

Immunohistochemistry (IHC) is based upon the specific detection of an antigen, using an antibody to recognize it and then staining the resulting complex. This is why it is also called immunostaining or immunodetection.

Selecting the Right Primary Antibody

A vital step in this procedure is choosing the right primary antibody. The appropriateness of the primary antibody is determined by the specificity with which it binds the target antigen.

Following this, binding the primary antibody is detected in one of two ways. Either directly, using a label conjugated to the primary antibody directly, or indirectly, using a secondary antibody which has already been labeled, to bind to the primary antibody which has formed a complex with the target antigen.

The secondary antibody can do this because it has been created to act against the host species, antibody type and subtype of the primary antibody. The detection is carried out through direct visualization of the fluorescent label, or by using an enzyme to act upon a soluble substrate, converting it into an insoluble chromogenic (color-generating) product.

Immunostaining Protocol

The following diagram illustrates a typical immunostaining protocol. When direct immunostaining is carried out, there is no need to incubate the primary antibody-antigen complex with the secondary antibody. When fluorescent labels are used, the last step, in which a soluble substrate is added, is left out.

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Last updated: Apr 1, 2019 at 5:40 AM

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