What is Immunohistochemistry?

 

Immunohistochemistry is a technique that uses antibodies conjugated to enzymes that catalyze reactions to form detectable compounds to visualize and localize specific antigens in a tissue sample. The root “histo-” specifically applies to biological tissue, so the process is only immunohistochemistry if it is being done in an organic tissue. In contrast, immunocytochemistry applies the same process to individual cells.

Performing Immunohistochemistry

Immunohistochemistry can be performed in a few steps. First, the tissue and cells must be “fixed” using a chemical like formaldehyde. This stabilizes the structural properties of cells, preventing them from changing throughout the process. Next, cells need to be permeated using a detergent such as Triton X, which allows antibodies to enter the tissue and bind to epitopes within the cell. Primary antibodies against a protein of interest are added, and secondary antibodies with enzymes like horseradish peroxidase (HRP) conjugated to their Fc domain are added to target the primary antibody. Enzymes like HRP can target certain substrate molecules like diaminobenzidine (DAB) and catalyze an oxidation which results in the creation of a colorful compound. This colorful compound will stay localized to the area the antibody was targeted to, staining the area near the protein of interest a different color from the rest of the tissue. Lastly, tissues are counter stained using a dye like hematoxylin to create contrast between the tissue stained using IHC and the non-colored regions for better visualization.

Purpose of Immunohistochemistry

IHC’s most important purpose is to be able to see where specific antigens are expressed in a tissue sample. Localization of specific proteins is important for diagnostics in fields such as cancer or infectious diseases.

For instance, IHC is often used in the diagnosis of certain forms of breast cancer. Overexpression of the HER2 receptor located in breast tissue is often correlated with breast cancer development. This is because the HER2 receptors receive signals from growth factors. These signals result in an intracellular signal transduction pathway ultimately leading to cell division and growth. Immunohistochemistry can be very helpful for detecting the amount of HER2 expression in a tissue biopsy. By targeting HER2 receptors with antibodies, localizing them with substrate stains and analyzing the levels of receptors in the tissue, it is possible to gather additional evidence about a patient’s current status and possibly diagnose them with a specific form of cancer.

Selecting Antibodies for IHC

In IHC there are primary and secondary antibodies used. Primary antibodies bind directly to the antigen, while secondary antibodies bind to the primary antibody. When selecting primary antibodies for IHC, there are three types of antibody preparations to choose from for IHC: polyclonal antibodies, monoclonal antibodies, or pooled monoclonal antibodies.

Polyclonal and monoclonal antibodies each have advantages and disadvantages:

  • Polyclonal antibodies result in greater staining and excellent signal, but can give false positives by binding unwanted sites.
  • Monoclonal antibodies have a high specificity, reducing the number of false positive bindings, but often hive a much weaker stain.
  • Pooled monoclonal antibodies also give excellent staining, as well as high specificity however there is limited availability for pooled monoclonal antibodies that do not bind noncompetitively.

For more information on Polyclonal and Monoclonal antibodies visit our blog.

For secondary antibodies, the antibody should be against the species the primary antibody was developed in. For example if the primary antibody is from a rabbit, the secondary antibody should be an anti-rabbit IgG, typically developed in another species like goats. Secondary antibodies will also be labeled with an enzyme like HRP, or biotin conjugated for staining or amplifying signal.

IHC is a popular technique for visualization in fields like cancer, neuroscience, and infectious diseases. If you can't find an antibody that fits your IHC needs, ProSci has over 20,000 catalog antibody products including primary and secondary antibodies, or consider starting a custom antibody project with us.

Sources:
https://www.breastcancer.org/symptoms/testing/types/ihc
https://www.prosci-inc.com/support/protocols/ihc-protocol/