How to select and optimize the primary antibodies for the IHC technique?

April 29, 2020 0 Comments

Immunohistochemistry (IHC) is a widely used technique to analyze the anatomy of the tissue of interest and to visualize the expression, location, and intensity of a specific antigen. Although IHC is a relatively easy and straightforward technique, the quality of staining can be influenced by variables that must be considered to produce reliable and consistent data. Next, we will indicate the concepts that you must take into account to select a primary antibody and perform the IHC technique successfully.


A primary antibody is an immunoglobulin that specifically binds to a particular protein or other biomolecule of interest for the purpose of purification, detection, and measurement. They are defined by their paratopes of the variable region of the antibody that show specific binding to the target epitopes of a protein. This specific binding transcends IHC, where antibodies can be used to detect specific antigen markers in tissue samples.

This binding force can often be quantified with the affinity constant (Ka). The factors that determine this value depend on how well the target epitopes fit into the antibody paratopes. It is of great importance to consider the binding affinity, since this property influences the considerations for the optimal dilution of the antibody, the incubation time and the resistance of the consecutive wash cycle in the IHC technique.


Information on the sequence of the target protein and post-translation states can usually be obtained from a protein sequence database such as UniProt. For many, the first validation method is to compare the homogeneity between the antibody immunogen and the sequence of the target protein using sequence alignment programs such as BLAST. In optimal situations, the use of additional techniques together can provide even greater validation for antibody-antigen compatibility.

A frequent problem is the possibility of a non-specific binding with other endogenous antigens that can be found in the sample. This often provides an undesirable background stain that can interfere with the results. However, it can be verified with the use of a negative control, which involves staining knockout tissue samples that do not express the target protein. A more accessible alternative would be to obtain an established cell line that does not express the target protein. In any situation, the expected results should show an absence of signaling, which should be compared with the tissue under study.

Another method to determine if there is sufficient reactivity of the primary antibody against the antigen is the verification with the Western Blot technique. Ideally, reactivity is represented by staining a single band located at the molecular weight of the target protein. Conversely, the presence of multiple bands near the desired molecular weight could indicate post-translational modifications or non-specific reactions.


Ideally, antibodies that have been validated for IHC should be used. Antibodies that have not been validated are often tested against at least one other non-immunohistochemical method to serve as sources of comparison for the success of the technique. However, it must be taken into account that an antibody that works successfully in a different protocol does not guarantee a good performance in IHC. A frequent example is Western transfer. Successful staining in Western Blot samples is a good sign that the antibody should also work in IHC.

However, the differences between these two procedures can also prevent success from being translated for the other methodology. For example, many IHC protocols frequently introduce binding reagents such as formaldehyde and glutaraldehyde. These compounds are capable of producing crosslinking bridges with proteins, causing small variations in the tertiary structures of proteins, which could cause the binding affinity of the antibody to its target epitope to decrease. This phenomenon may not be observed in the Wester Blot technique, where cell lysates or homogenates can be used, where the proteins are in their native form. However, the validation of these non-immunohsitochemical methods, serve as invaluable tools to provide information on experimental success.


In general, caution should be used when using an antibody that has been produced in the same host where the study is to be performed. Staining with secondary antibodies is generally directed against the species where the primary antibody was produced. If the samples include the same animal species, both in the primary antibody and in the tissue under study, it can be expected that the secondary antibodies indiscriminately stain all the structures in the samples, which would cause an error in the interpretation of the results.

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