For its easy and rather inexpensive use in the search for tissue antigens that range from amino acids and proteins to infectious agents and specific cellular populations, immunohistochemistry is an essential tool in the diagnostic routine of pathological anatomy laboratories.

In the last couple of decades, there has been an exponential increase in publications on immunohistochemistry techniques spanning cellular and molecular biology, biochemistry, pathology, histology, immunology, internal medicine, and surgery scientific articles.

Today we meet with Dr. Caroline Kampf to ask her 10 questions about the past, present, and future of immunohistochemistry (IHC).

Caroline is the Chief Scientific Officer (CSO) and a co-founder of Atlas Antibodies AB, Sweden. Dr. Kampf is Associate Professor and has a Ph.D. in Cell Biology from Uppsala University. For over ten years Caroline played a key role in starting the Human Protein Atlas project where she most recently held the position as Site Director for the Uppsala site, responsible for the immunohistochemical antibody validation. Prior to joining Atlas Antibodies, she worked at Olink Proteomics AB and Affibody AB.

Photo: Dr. Caroline Kampf, Chief Scientific Officer (CSO) and a co-founder of Atlas Antibodies AB.

Q1: Caroline, could you explain IHC in simple words?
Immunohistochemistry, or IHC, is a powerful method enabling the visual detection of antigens, mostly proteins in cells and tissues. Visualization of the antibody-antigen complex is generally done with conjugated secondary antibodies (eg peroxidase or fluorochrome tagged) and includes a detection step where the interaction is visualized. IHC is routinely used in the field of diagnostics and basic research.

Q2: What does it deliver in terms of biological insights?
Protein expression in its histological context on a cellular level. By counterstaining the tissues or cells you get a high contextual resolution context whereby the visualized proteins are interrogated in relation to other non-stained cells. The information on the protein distribution at the cellular level and comparative differences in this expression pattern is essential for pathologists when diagnosing tumor specimens (tumor diagnostics).

Q3: What is the translational application?
Traditional IHC is based on the immunostaining of thin sections of tissues attached to individual glass slides. The progress in the field of IHC-based techniques and reagents has enabled scientists and healthcare providers with more precise tools, assays, and biomarkers.

There are several ways you can use IHC. In diagnostics and clinical practice, physicians can investigate protein expression by comparing healthy and diseased material looking for relevant biomarkers helping the pathologist to estimate the therapeutic outcome.

Basic research can investigate relationships, pathways, etc. Working with unknown targets or poorly characterized proteins several important efforts are contributing to a complete map of protein expression.

The Human Protein Atlas (HPA) project is a prime example of how high-throughput IHC is used to achieve large-scale mapping of the human proteome in a multitude of tissues, cancers, and cells.

Q4: Share a turning point or defining moment in IHC history.
Immunohistochemistry is a routinely established and rather old method, first documented for use in the early 1940s. Many years of technical development and the increased accessibility of specific affinity reagents have greatly improved the usefulness and areas of applications for IHC. There is an interesting paper to read if you are curious about the history of immunohistochemistry.

Q5: How prevalent is the use of IHC today?
Immunohistochemistry is widely used in both research and clinical practice and the method can quickly be performed in most laboratories. The procedure is short, simple, and cost-effective.  

According to Pubmed, the database of peer-reviewed and published scientific articles, in the last five years IHC has been used in 10,641 research studies. This means on average more than 2000 papers per year, the equivalent of 5 papers per day! It is quite impressive for a rather old technique. 

Q6: What do you see as a barrier to the wider adoption of IHC?
This technique relies on specific and reproducible antibodies. The lack of standardized guidelines for determining the specificity and functionality of antibodies renders the translation of promising biomarkers to the clinic difficult. Often pathologists use a panel of several antibodies to helpfully classify a tumor.

We at Atlas Antibodies are a step ahead for the systematic validation of antibodies. We are the original manufacturer and the provider of enhanced validated antibodies targeting all human proteins. All our antibodies are rigorously validated for specificity and reproducibility and are characterized in several applications.

Q7: Neuroscience and cancer research: how IHC enhances understanding of diseases?
Well, one example is the evaluation of Human Epidermal Growth Factor Receptor 2 (HER2) in breast cancer. HER2 is over-expressed in around 20–30% of breast cancer tumors.

By using IHC and an antibody against the HER2 receptor, the localization of the receptor and the level of receptors could guide you to the current status of the patient and possible breast cancer diagnosis and outcome.

Another great example is the detection of beta-amyloid, extracellular filamentous protein deposits in the brain that can be used for the diagnosis of Alzheimer's disease.

Q8: What questions were you addressing when you started using IHC?
I was investigating a chloride channel important for airway hypersensitivity and got an in-house developed antibody from a collaborator in the early nineties. We wanted to localize the exact localization of this protein and the way the expression changed after different stimuli.

By counterstaining, the tissue investigated we obtained a high-resolution image of the entire airway epithelium and included cell types and the distribution of the chloride channel. After that, my experience is through the Human Protein Atlas project, producing antibodies towards all human proteins, and screening their localization in normal and cancer tissues.

Q9: What happens next in the process of IHC and discovery?
Today, technological advancements, high-throughput and automated systems, and highly validated reagents allow IHC to be performed faster, with greater sensitivity, simultaneously querying multiple and difficult markers in more quantitative and automated ways. The future is indeed bright for IHC.

Q10: If you could offer readers interested in IHC one key piece of advice, what would it be?
Use validated antibodies and optimize your protocol thoroughly.