Careful planning and the fine tuning of experimental protocols are key to ensuring clear, interpretable scientific results. This is especially true for immunohistochemistry (IHC) studies, where each step in the often multiday process – from tissue preparation to stain development – can significantly impact the final outcome and analysis. Often, the simultaneous examination of multiple antigens is required to address specific scientific questions, which further complicates IHC protocol development. A general understating of the steps necessary to optimize IHC for multiple targets is essential to achieve reliable results. So, what are these steps?
Before we outline guidelines for dual staining, let’s first review the basic principles of immunohistochemistry. IHC has long been used as an important diagnostic tool in medicine as well as an investigative tool in basic research. IHC is an “information rich’’ procedure, in that it allows scientists to not only determine whether a target of interest is present, but also to see where it is expressed in the native histological context of the sample. By layering stains for multiple antigens, IHC has the potential to provide novel insight into underlying biological processes or distinguish between normal and pathological states.
IHC works by exploiting specific antibody-antigen interactions paired with a detection method to produce a reaction that is visible by microscopy. Cellular proteins of interest are the most common antigens investigated, but carbohydrates and, in some cases, nucleic acids can also be examined by IHC.
The most important component of any IHC experiment, aside from the tissue preparation itself, is the primary antibody, as it directly influences the specificity of IHC reactions. Primary antibodies used in IHC come in two forms, polyclonal and monoclonal. Polyclonal refers to a collection of antibodies raised in the same host that recognize multiple regions, or epitopes, of a target antigen. Monoclonal, on the other hand, denotes a single antibody that binds to one specific epitope. Each class of antibodies can be manufactured by immunizing a variety of hosts – from rabbits to goats – with the desired antigen, followed by purification directly from serum or by the isolation of antibody-producing B-cells for hybridoma development.
The antibody-antigen interaction can be detected by a number of methods. One option is the direct conjugation of a fluorescent tag or chromogenic enzyme, like horseradish peroxidase (HRP), to the primary antibody. However, a more common approach is the use of tagged secondary antibodies, which recognize and bind to the primary antibody backbone, or heavy chains, in a species-specific manner. The use of secondary antibodies offers several advantages over direct conjugation, including signal amplification and the cost-saving flexibility of using them in combination with multiple primary antibodies. Finally, independent chromogenic or fluorescent tags can be used for multicolor detection of several primary/secondary antibody pairs in the same tissue sample.
This brings us back to our initial question: What are the steps necessary to optimize the simultaneous detection of two antigens by IHC? First, validated primary antibodies to targets of interest – ideally produced in separate host species – should be selected and combined with species specific secondary antibodies for detection. This general rule eliminates the concern for cross-reactivity during staining. Next, the staining parameters for each primary antibody must be tested in parallel. This includes determining the appropriate antibody concentration, incubation time and temperature, and chromogen pairing to produce results with the sharpest specificity, while minimizing background staining. Once the conditions for individual antibodies have been established, the optimal order of stain development must be evaluated to account for differences in chromogen reactivity. Finally, results of the completed dual stains should be compared to initial single stain tests. If necessary, chromogen development times should be adjusted in order to achieve balance between signals. These steps can be adapted for any number of target antigens and tissues samples, and their use ensures confidence in the final interpretation of dual staining by IHC.
For more details on this procedure, we encourage you to refer to our recent application note: Optimizing Immunohistochemistry Dual Staining to Visualize and Understand Protein Co-expression during EMT.