Direct and indirect detection

Successful immunostaining relies on the primary antibody specifically binding to the target antigen. The antibody is then detected either directly, through a label conjugated to the primary antibody, or indirectly, using a labeled secondary antibody.

Direct detection is ideal for detecting highly expressed antigens and doesn’t require an additional incubation step with a secondary reagent. Indirect detection is more suitable for studies of poorly expressed antigens, which benefit from the signal amplification provided by the secondary reagent.

Signal amplification occurs through the potential for two or more labeled secondary antibodies to bind to each primary antibody. The signal may be amplified further by using avidin or streptavidin with biotinylated secondary antibodies. However, the use of a secondary antibody requires additional blocking steps and controls.

Choosing and optimizing primary antibodies for IHC

As the choice of primary antibody is critical to the success of the IHC experiment, it is essential to consider these factors.

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

​The specificity of the antibody is usually determined experimentally. Staining patterns consistent with the known localization of the target protein in control cells or tissues can indicate specificity.

Comparison of the immunogen sequence to other proteins using alignment tools such as BLAST may also indicate antibody specificity, but it is not conclusive.

The most definitive demonstration of antibody specificity is the lack of staining in tissues or cells where the target protein has been knocked out.

Has the antibody been proven to work in IHC?

Antibodies are validated for distinct techniques due to differences in antigen recognition. For example, western blotting experiments are run under denaturing conditions, whereas the native (3D) form of the protein is more likely to be preserved in IHC. Consequently, an antibody that has been validated for western blot may not work in IHC.

It is important to note that fixation and antigen retrieval methods also significantly impact the ability of an antibody to recognize the epitope of interest in an IHC experiment.

The method of antibody manufacture

There are three main methods of antibody manufacture – serum-purified polyclonal, hybridoma-derived monoclonal, and recombinant. The type of antibody manufacture can significantly impact your results, so it is essential to understand the different methods:

• Serum-purified polyclonal antibodies are a heterogeneous mix of antibodies obtained by immunizing an animal with the antigen of interest.
• Hybridoma-derived monoclonal antibodies are produced from a single-clone antibody-producing B-cell that is fused to a myeloma.
• Recombinantly manufactured antibodies are produced recombinantly by cloning antibody-coding genes into high-yield expression vectors.

Which host species should be used for the primary antibody?

Ideally, the primary antibody should be raised in a host species different from the sample species to avoid cross-reactivity with endogenous immunoglobulins in the tissue. If you are using a mouse primary antibody on mouse tissue, specialized mouse-on-mouse IHC kits are available.

Antibody optimization

The quality of staining is influenced by many variables, including the primary antibody concentration, the diluent used, the incubation time, and temperature. Optimization may be required for each antibody and sample in order to achieve specific staining with minimal background. To gain optimal staining:

• Vary antibody concentration while maintaining a constant incubation time and temperature.
• Try a longer incubation time to help the antibody penetrate the tissue
• Combine longer incubation times with lower temperatures to promote specific binding, eg instead of 1 h at room temperature, try overnight at 4°C

Secondary antibodies in IHC

To ameliorate non-specific binding, the secondary antibody must be directed at the species in which the primary antibody was raised but must itself be raised in a different species.

For example, if the primary antibody is raised in rabbit, an anti-rabbit secondary antibody raised in a species other than rabbit must be used. It is also important that the isotype that the secondary antibody is raised against matches the primary antibody’s isotype.

As with primary antibodies, there are considerations for the optimal secondary antibody.

Pre-adsorption of secondary antibodies

Pre-adsorption is an extra purification step to increase the specificity of a secondary antibody. The secondary antibody is passed through a column matrix containing immobilized serum proteins to separate the highly specific antibodies from the non-specific antibodies.

F(ab’)2 fragment secondary antibodies

F(ab’)₂ fragment secondary antibodies are recommended for staining tissues that are rich in Fc receptors (eg spleen, thymus, blood). F(ab’)₂ fragment secondary antibodies, which are smaller and penetrate tissues more efficiently, are beneficial for multiple IHC staining.

HRP-polymer secondary antibodies for low-expressing proteins

For higher sensitivity, horseradish peroxidase (HRP)-polymer secondary antibodies use micropolymer technology to form smaller detection complexes that allow improved tissue penetration and sensitivity. In addition, HRP-polymer secondaries bind more HRP than standard HRP secondary antibodies, increasing signal.