Immunohistochemistry (IHC) is a widely applied experimental method used to examine tissue antigen expression and behavior with the use of an antibody conjugated to a secondary tag for visualization. IHC consists of a tissue preparation phase, an antibody-staining phase, and a result analysis phase - all of which may lead to skewed results if not properly performed. One way to ensure that IHC staining results are in fact demonstrating protein behavior and not a side effect the experimental process is to use IHC controls within each experiment. By doing so, you can feel confident in your protein integrity, experimental methods and primary and secondary antibody efficacy.
To begin, the manner in which tissue is harvested, dissected and mounted to a microscope slide is very important. It is vital that the tissue is collected rapidly in order for the proteins to stay in a stable state. Soon after tissue collection comes the fixation step of IHC using a fixative, which is most commonly the fixative formaldehyde. Depending on the protein’s reaction to the fixative, it is wise to perform a few different fixation lengths with varying percentages of fixative using your target antibody to determine which protocol produces the cleanest results. If the tissue is fixed for too long of a time period, the covalent crosslinking effect of the fixative results in an ability for the antigen specific antibody to effectively bind or an overwhelming amount of non-specific protein binding. Yet, under-fixation can result in protein degradation and inability to bind. There are also other forms of fixatives available that may be more appropriate to your tissue sample or protein of interest, including acetone or ethanol.
The same principle applies to the permeabilization phase of the IHC protocol, where a mild detergent is introduced to the tissue in order to perforate the cell membrane in order to allow the entry of the primary antibody. Over permeabilization can result in an abundance of background staining in your primary antibody stain, and under permeabilization can result in no binding of your primary antibody to your antigen.
Once you are prepared to apply a primary antibody to your tissue, it is important to use a control tissue sample when available. For example, staining a tissue that is not known to express the protein of interest will help to confirm unspecific binding of the antibody. Another way to achieve primary antibody staining efficiency is to add normal serum in place of the primary antibody to see the effect it has on the secondary antibody binding. Both primary and secondary antibodies need a level of optimization within IHC to ensure there isn’t over saturating of an antibody resulting in false positives. Using a secondary antibody only (no primary antibody control) should produce little to no visual background, which is an important control to apply. Lastly, using an isotype control is important to confirm that the primary antibody is binding to a specific antigen and not the immunoglobulin of the tissue.
Learn more about the importance of IHC controls
Matos LL, Trufelli DC, de Matos MG, da Silva Pinhal MA. Immunohistochemistry as an important tool in biomarkers detection and clinical practice [PMID: 20212918]
