After a few intense days of meticulous tissue washes and antibody incubations, my staining is finally ready. I am staring at it down the microscope. I enjoy what I see: a dark brown color exactly where I expect to see it! Am I good or what? I give myself a figurative pat on the back and an imaginary high-five. Excited and proud, I call my supervisor, who glares intently at it and then firmly, and rather surprisingly, asks me to re-run the staining. What went wrong? What did I miss? I must get to the bottom of this!

Immunohistochemistry (IHC) is a common assay in scientific research and a precious resource in pathology for morphologic diagnosis and for studying the pathogenesis of the disease.

Proper methodology and interpretation of an immunohistochemical assay are absolutely vital. Besides finding a specific antibody, a major challenge in IHC is that of reducing non-specific interactions without, at the same time, impairing antibody-epitope binding.

Yet, it takes multiple efforts and deep scientific knowledge of the technique, the tissue, and the target protein to discriminate false positives.

Good immunohistochemical staining is achieved when a sufficient quantity of primary antibody penetrates the sample to bind its related antigenic target with high specificity. However, in most cases, the same physiochemical forces that govern specific antibody-antigen interactions, such as hydrophobic interactions, ionic interactions, and hydrogen binding, also contribute to non-specific binding and other artifacts.

3 commons artifacts in immunohistochemistry

Commons artifacts in immunohistochemistry include non-specific binding, high background, overstaining, or too-weak staining.

Nonspecific binding refers to the binding of the antibody, either the primary or the secondary antibody, to something else in the tissue than its designated target, such as other proteins or different epitopes in the target protein.

Overstaining (high background) occurs when the level of background becomes so high that it obscures important features and structures of the tissue. 

Weak staining could be defined as a staining of moderate intensity present in no more than 10% of cells.

Immunohistochemistry troubleshooting: how do I solve my case? 

I love and admire the uncompromising detective work of collecting clues that’s always central to a criminal investigation. Crime scene investigation is the meeting point of science, logic, and rigorous protocols. Observe, record findings, interview suspects, and gather facts and evidence related to suspects. And you could claim that identifying the source of error in immunohistochemical staining is an identical exercise.

It is the role of the detective to examine all the physical evidence and to secure and investigate the scene of the crime. Processing a crime scene is a long, painstaking process involving detailed documentation of the conditions at the scene and the collection of any physical evidence that could possibly help to reveal what happened, why it happened, and point without a shadow of a doubt to the culprit. 

Apply the same approach to your immunohistochemistry troubleshooting.

Looking at my staining: what do I see?

For any investigation, the details of events provided by witnesses are a critical element of the evidence gathered. In an immunohistochemistry experiment, the accurate analysis of the staining results will provide insights from different perspectives, but these perspectives need to be carefully assessed to establish the reliability of the evidence provided the cause, and the required solution.

  

The usual suspects

An immunohistochemistry experiment is a combination of 3 major elements: sample, application, and experimental protocol. An excellent IHC staining is achieved when all the required elements, qualities, and characteristics are matched perfectly. A combination as good as it is possible can be.

So, if you are looking for suspects to blame for artifacts, keep in mind the 3 usual suspects:

• Sample (tissue or cell)
• Antibodies (primary and secondary)
• IHC protocol

Look carefully at the table below: these tips will help you to identify the possible culprit.  

TABLE

OK, you have an idea now what to blame for the artifacts in your IHC staining. That sounds good enough, but when it comes time to do, to solve those artifacts, it is hard to know where to start. 

Readings

Buchwalow I. et al. Sci Rep. (2011) 1: 28, Non-specific binding of antibodies in immunohistochemistry: fallacies and facts.

De Matos L.L., et al. Biomarker Insights (2010):5 9–20, Immunohistochemistry as an Important Tool in Biomarkers Detection and clinical practice.

Ramos-Vara J.A. and Miller M. A. Veterinary Pathology (2014) 51(1) 42-87, When tissue antigens and antibodies get along: revisiting the technical aspects of immunohistochemistry: the red, brown, and blue technique.

Ward J.M. and Rehg J.E. Veterinary Pathology, (2013) 51 (1) 88-101, Rodent Immunohistochemistry: Pitfalls and Troubleshooting.