IHC guide v3 952x200px

Choosing and optimizing primary antibodies for IHC

Learn how to select and optimize the correct primary antibody when designing your immunohistochemistry (IHC) experiment.

This is driven by the specificity of the primary antibody for the epitope of interest.  However, a number of other factors must also be considered when choosing the primary antibody.

Is the antibody specific for the protein/epitope of interest? 

Is the antibody specific for the protein/epitope of interest? 

The specificity of the antibody is usually determined experimentally. Comparison of the immunogen sequence to other proteins using alignment tools such as BLAST may give some indication about antibody specificity but is not conclusive. The most conclusive demonstration of antibody specificity is lack of staining in tissues or cells in which the target protein has been knocked out. Other indicators are recognition of a single band in western blotting (see below for note on why this is not very reliable) and staining patterns that are consistent with known localization of the protein of interest in control cells or tissues. Ideally, the antibody should recognize the target antigen in the species of interest. If this is not the case, sequence comparison of the immunogen with the corresponding region in the protein from the species of interest may give some indication of specificity. 

Has the antibody been proven to work in IHC? 

Antibodies that recognize the target proteins in western blotting experiments, which are run under denaturing conditions, may not always recognize the antigen during in IHC, where the native (3D) form of the protein is antigen is more likely to be preserved. An antibody that has been shown to work in IHC or immunocytochemistry (ICC) is preferable. It is important to note that fixation and antigen retrieval methods also have significant impact on the ability of an antibody to recognize the epitope of interest in an IHC experiment.

Monoclonal or polyclonal?

As monoclonal antibodies are produced from a single B cell clone, they represent a homogeneous population that binds to a single epitope. As a result, they are less likely to cross-react with other proteins (provided that the clone recognizes a unique epitope) and therefore produce less background staining than polyclonal antibodies. The production of monoclonals from a hybridoma also means that there is less variation between different antibody lots than polyclonal antibodies. 

Polyclonal antibodies are heterogeneous populations that can recognize multiple epitopes. They are therefore more tolerant of changes in protein conformation (resulting, for example, from fixation or changes in temperature). They are also more stable over a range of pH and salt concentrations than monoclonal antibodies. 

Which host species?

Ideally, the primary antibody should be raised in a host species that is different to the species of the sample in order to avoid cross-reactivity with endogenous immunoglobulins in the tissue. 

Antibody optimization

The quality of staining is influenced by the primary antibody concentration, the diluent used, the incubation time and temperature. All of these variables may need to be optimized for each antibody and sample in order to achieve specific staining with minimal background. Usually, antibody concentration is varied while maintaining a constant incubation time and temperature in order to optimize staining. Longer incubation times may be used to ensure that the antibody penetrates the tissue. Longer incubation times can be combined with lower temperatures to promote specific binding.

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