Considerations for immunohistochemistry

Balaji, S., Li, H., Steen, E. et al.

Although immunohistochemistry (IHC) has been known and applied for decades, major advances such as robotic sample processing, digital slide image capture, and innovative computerized data analysis uphold the relevance of the technology in current laboratory practice. In keeping with this notion, the expression of a protein by a given tissue or cell can be readily defined using methods like immunoblotting or mass spectrometry. However, when it comes to assessing the microenvironmental location and potential cellular connections, only IHC can provide the pathophysiological spatial context. Thus, IHC enables the selective localization and visualization of protein antigens in tissue sections by means of antigen-specific antibodies that are conjugated to selective fluorescent or enzymatic tags, which can be readily revealed by fluorescence microscopy or chemically pigmented reactions. Since IHC relies on the highly specific antigen and antibody interactions, the technology can be used to identify cell or tissue antigens that range from amino acids and proteins to infectious agents and specific cellular populations and their relevant functional properties, thus underscoring its critical application as a key research/diagnostic tool for investigators studying animal or human tissues. With well-developed tests and controls, correct procedure, and equipment, IHC can be used to analyze a wide variety of cell and tissue structures, processes, and functions, such as cell cycle analysis and tissue protein binding in health and disease. Notably, these applications were previously inaccessible by traditional histochemistry staining techniques, which only identified a limited number of proteins and tissue structures. In the past, this process was semiquantitative at best, but new advances are making it possible to obtain quantitative results using IHC. The present chapter reviews the history and applications of IHC, basic principles, techniques, troubleshooting, analysis, and data interpretation. This chapter will further cover the latest innovations in IHC slide preparation and labeling for advanced diagnostic and prognostic applications, such as CyTOF and laser capture microdissection, which allows high-throughput quantitative methods of proteomics, real-time polymerase chain reaction (qRT-PCR), and genomics on selective cells and tissue regions.


Balaji, S., Li, H., Steen, E. et al. "Considerations for immunohistochemistry" Success in Academic Surgery: Basic Science (2019): 105-144