Immunohistochemistry (IHC) is a powerful technique widely used in the field of biology and medicine to visualize and study the distribution, localization, and abundance of specific antigens within tissues. This technique relies on the use of monoclonal and polyclonal antibodies, making it an indispensable tool for researchers exploring various aspects of cellular biology, pathology, and disease mechanisms.1
In IHC, antibodies are used as molecular tools to recognize and bind to the protein of interest within cells and tissue sections, typically examined with a light microscope. There are two main types of antibodies used in IHC:
Primary Antibodies: These are specifically designed to recognize and bind to the target antigen and selected based on their specificity and affinity.
Secondary Antibodies: These are labeled with reporter molecules such as fluorophores, enzymes, radioactive elements, or colloidal gold. They do not directly target the antigen but bind to the primary antibody, enabling the visualization of the antigen-antibody complex. This amplification step enhances the sensitivity of IHC.
Workflow
Sample Collection and Preparation: Tissue samples are collected, fixed, and embedded in paraffin or frozen in optimal cutting temperature compound.
Antigen Retrieval: Formalin fixation can mask antigenic epitopes. Heat-induced antigen retrieval methods, such as microwave or pressure cooking, are often used to unmask and expose these epitopes.
Blocking: To reduce non-specific binding, tissue sections are treated with blocking agents, such as serum or bovine serum albumin.
Primary Antibody Incubation: Tissue sections are incubated with the primary antibody to allow for antigen-antibody binding, and excess primary antibody is washed away to minimize background staining.
Secondary Antibody Incubation: Incubation with a secondary antibody conjugated to a reporter molecule forms a complex with the primary antibody, allowing for signal detection. Washing is again performed.
Signal Development: Enzyme-linked secondary antibodies can react with substrate solutions to produce a colored precipitate, while fluorescently labeled secondary antibodies emit fluorescence when exposed to appropriate wavelengths.
Counterstaining: Counterstains like hematoxylin can be used to visualize cellular structures. The stained sections are then mounted on slides for analysis.
Imaging: Finally, the tissue sections are examined under a microscope, and the images are analyzed to determine the localization and abundance of the target protein.
Applications
Immunohistochemistry is a versatile technique with a wide range of applications.
Cancer research: IHC helps identify cancer biomarkers and characterize tumor tissues, aiding in diagnosis and treatment decisions. This is because specific tumor antigens are expressed de novo or are upregulated depending on the type of cancer, but IHC can also detect enzymes, oncogenes, tumor suppressor genes, and tumor cell proliferation markers.2
Neuroscience: Researchers can use IHC to study the distribution of specific proteins in the brain and nervous system. One study by Delcambre et al. used IHC to identify antibodies to detect neural and inflammatory cells in equine brain tissue, since there are a limited number of available species-specific reagents that react with formalin-fixed, paraffin embedded tissue.3
Pathology: In pathology, IHC is used in routine diagnostic work and basic or clinical research. For instance, researching biomarkers with IHC allows for the confirmation of target molecule expressions in the context of microenvironments.4
Drug Development: IHC plays a role in drug target identification and validation, as well as in assessing treatment efficacy. It can be used in all steps of a clinical trial, particularly in image analysis which is important in drug efficacy, safety, target validation, and biomarker expressions.
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References:
Magaki, S., Hojat, S. A., Wei, B., So, A., & Yong, W. H. (2019). An Introduction to the Performance of Immunohistochemistry. Methods in Molecular Biology (Clifton, N.J.), 1897, 289. https://doi.org/10.1007/978-1-4939-8935-5_25
Duraiyan, J., Govindarajan, R., Kaliyappan, K., & Palanisamy, M. (2012). Applications of immunohistochemistry. Journal of Pharmacy & Bioallied Sciences, 4(Suppl 2), S307. https://doi.org/10.4103/0975-7406.100281
Delcambre, G. H., Liu, J., Herrington, J. M., Vallario, K., & Long, M. T. (2016). Immunohistochemistry for the detection of neural and inflammatory cells in equine brain tissue. PeerJ, 4. https://doi.org/10.7717/peerj.1601
Kim, W., Roh, J., & Park, S. (2016). Immunohistochemistry for Pathologists: Protocols, Pitfalls, and Tips. Journal of Pathology and Translational Medicine, 50(6), 411-418. https://doi.org/10.4132/jptm.2016.08.08
