The Art of Immunohistochemistry

Immunohistochemistry is a method that is used to detect antigens in tissue sections and cells using antibodies. It is a reaction that is based on antigens and antibodies of a tissue. There are two ways that I use IHC for drug development. The first is IHC detection of a common CD marker to identify a particular cell type in a tissue. The second use of IHC is for what is referred to as tissue cross reactivity. In tissue cross reactivity, the antibody (i.e. drug) is put on naive tissue and then detected so that we can see where the antibody is binding under a microscope. We want the antibody to bind to its target without binding to anything else. By identifying the target and engineering the antibody to target it, we are able to develop medications that will serve a specific purpose based on the epitope of the protein that it is meant to bind to.

http://arstechnica.com/science/2011/11/light-kills-tumors-that-are-first-targeted-with-a-photosensitive-agent/
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The tissue is sectioned on a cryostat or microtome and can be fresh frozen or paraffin embedded. There are different methods  that are needed for paraffin tissues compared to fresh frozen tissues. For successful IHC staining of paraffin embedded tissues, an antigen retrieval step is needed in the assay. Some common ways we perform antigen retrieval include heat induced retrieval, and the application of proteolytic enzyme solutions such as Trypsin or Proteinase K. Fixation is important to preserve the morphology and target proteins in the tissue. Paraformaldehyde is a common fixative, however, acetone, methanol, and a combination of acetone and paraforamlehyde are also used. It is crucial to test multiple fixations to select the most appropriate condition for the tissue and the target proteins.

We need to quench endogenous peroxidase activity because the enzymes peroxidase and alkaline phosphatase are often used in IHC to convert the colorless chromagens into colored end products for detection of antibody binding. The idea is to detect the antibody binding with minimal background staining. Common causes of increased background could indicate that the concentration of the antibody is too high so it is important to test multiple concentrations to select the optimal concentration to use for the assay. Endogenous avidin binding activity can also lead to increased background staining which can be blocked by treating the tissue with biotin. Sometimes polymer-based detection methods can also cause background staining which can be decreased by increasing the quantity and time period for wash steps between reagent treatments.

Depending on the primary antibody (i.e. the drug or CD marker), a secondary and possibly a tertiary antibody is needed. Think of it as building a ladder to amplify the signal of where the antibody is binding while blocking all of the background noise so that we can see what is happening. There are many reagents available to develop the method for an IHC assay, so there are multiple methods, reagents, concentrations, and incubation times that can render the same result. We find the best method for detection of the antibody within the assay and then identify target and non-target binding of the antibody to determine the safety and effectiveness of the drug.iPhone June2015 195

A pathologist is able to analyze the slides to identify any binding of the antibody to the tissues. The staining on the tissues can be quite remarkable. Seeing the results of an IHC stain is like looking at a painting once it is complete. Instead of brushes and paint we are using reagents and antibodies. The tools we use in the laboratory work together to create a window into what is happening inside of our bodies. As simple as it sounds to cure disease or make medicine, it is much more complex just as our bodies are indefinitely complex. By building on the knowledge of those before us and continuing research we will advance medical technology and have the capability to save more lives than we ever thought possible.

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