IHC: Considerations for Antibody Selection & Multiplexing

Immunohistochemistry (IHC) is a technique used for visualizing tissue sections. Unlike other immunoassay techniques, it offers insights into protein distribution and relative abundance that can differ in conditions of health and disease.

Antigen retrieval is the process of removing epitope-masking protein cross-links formed during formalin fixation. It is achieved via one of two methods - heat-induced antigen retrieval (HIER), which uses heat and an optimized buffer, or proteolytic antigen retrieval (PIER), which relies on enzymes such as proteinase K, trypsin, and pepsin. HIER is usually preferred due to being more controllable, but it is important to check antibody datasheets for the recommended antigen retrieval protocol.

A main consideration for IHC is whether to use monoclonal or polyclonal antibody reagents, or a combination of both. Monoclonals are highly specific, typically demonstrate consistent lot-to-lot performance, and are available in unlimited supply. Polyclonals recognize multiple epitopes and can provide signal amplification to aid the detection of scarce analytes. Recently, recombinant monoclonals have become increasingly popular since they address the issue of genetic drift that can affect antibody performance over time; they also provide ample opportunities for modification and engineering.

It is equally important to choose between direct or indirect detection. Direct detection uses labeled primary antibodies and offers the advantages of fewer protocol steps and greater flexibility for multiplexing. Indirect detection instead relies on labeled secondary antibodies, which provide signal amplification. For indirect detection, it is critical that cross-adsorbed secondary antibodies are used to avoid non-specific antibody binding to the sample.

Whichever immunostaining method is chosen, it is essential to select antibodies that have been validated for IHC and proven to specifically detect the target of interest. This information should be available on antibody datasheets, which should additionally state whether the validation was performed using frozen or FFPE tissue.

When performing multiplexed IHC, careful antibody selection is crucial to ensure reagents will work in combination. For example, antibodies against cell surface proteins may be incompatible with staining protocols used for detecting intracellular markers, which involve fixation and permeabilization, while panel design is vital to avoid spectral overlap between different fluorophores. Experimental controls are also key and can be selected by referring to open-access resources such as UniProt, PAXdb, or proteinatlas.org for information about expected protein expression. To circumvent the need for laborious optimization, preformatted antibody panels are a cost-effective solution that can often save time and usually lead to stronger signals.