Immunohistochemistry: A Guide to Protocol and Applications

INTRODUCTION

Immunoassays are laboratory techniques that use antibodies to detect and measure the presence of specific molecules in a sample. They are based on the principle of the immune system, which can recognize and bind to specific molecules, called antigens, with high specificity and affinity. Immunoassays have revolutionized the field of diagnostics and biomedical research, allowing for the detection of a wide range of analytes, including proteins, hormones, drugs, and infectious agents.

Here is a brief timeline of the major developments in immunoassay techniques;

• 1941: The first radioimmunoassay (RIA) is developed by Dr. Samuel Yalow and Dr. Solomon Berson.

• 1960s: Enzyme-linked immunosorbent assays (ELISAs) are developed by several researchers, including Rosalyn Yalow and Solomon Berson, Peter Perlmann and Eva Engvall, and Anton Schuurs and Bauke van Weemen.

• 1971: Fluorescence immunoassays (FIAs) are developed by researchers at the University of Amsterdam, including Anton Schuurs and Bauke van Weemen.

• 1975: Immunoradiometric assays (IRMAs) are developed by Yves Christol and Michel Aubert at the Atomic Energy Commission in France.

• 1980: Chemiluminescent immunoassays (CLIAs) are developed by Roger Y. Tsien and coworkers.

• 1981: Immunohistochemistry (IHC) is developed by Albert Coons and coworkers.

• 1985: Flow cytometry-based immunoassays are developed by Leonard A. Herzenberg and coworkers.

• 1990s: Multiplex immunoassays, which allow for the simultaneous detection of multiple analytes, are developed by several groups of researchers, including Ray Houghton and coworkers at Luminex Corporation and David R. Walt and coworkers at Tufts University.

Immunohistochemistry (IHC) is a type of immunoassay that is used to visualize the distribution and localization of specific proteins in tissue samples. It was first developed by Albert Coons and coworkers in 1981, who used fluorescently-labeled antibodies to detect antigens in frozen tissue sections. IHC has since become a widely used technique in pathology and biomedical research, allowing for the identification and characterization of cellular and tissue markers in health and disease. It can be used for a wide range of applications, including cancer diagnosis, classification, and prognosis, as well as for the identification of infectious agents and the study of immune responses.

---

TYPES

There are several types of immunohistochemistry (IHC) that can be used to detect and visualize specific proteins or other biomolecules within tissues. Here are some common types of IHC:

1. Direct IHC: In direct IHC, a single antibody is used to directly label the target antigen. The antibody is conjugated to a detection system, such as a fluorescent dye or an enzyme, which allows visualization of the target antigen. Direct IHC can be used to detect and visualize proteins that are expressed at high levels in the tissue, such as cytoskeletal proteins or structural proteins. For example, direct IHC can be used to visualize the distribution of actin or tubulin in cells or tissues.

2. Indirect IHC: In indirect IHC, a primary antibody is used to bind to the target antigen, followed by the addition of a secondary antibody that is conjugated to a detection system. This amplifies the signal and allows for more sensitive detection of the target antigen. Indirect IHC is more sensitive than direct IHC and can be used to detect proteins that are expressed at low levels in the tissue or are difficult to detect with direct labeling. For example, indirect IHC can be used to visualize the distribution of growth factors or cytokines in tissues.

3. Sandwich IHC: In sandwich IHC, two antibodies are used to detect the target antigen. The first antibody binds to one site on the antigen, and the second antibody binds to a different site on the antigen, allowing for detection of the antigen in a sandwich-like manner. Sandwich IHC can be used to detect and visualize proteins that are present at low levels in the tissue or are difficult to detect with other types of IHC. For example, sandwich IHC can be used to detect and quantify the levels of specific receptors or signaling molecules in tissues.

4. Competition IHC: In competition IHC, a known amount of the target antigen is added to the tissue section prior to the addition of the primary antibody. The primary antibody then competes with the added antigen for binding sites on the tissue section, allowing for quantitative analysis of the target antigen. Competition IHC can be used to quantify the levels of specific proteins in tissues and can be used to compare the levels of the same protein in different tissue samples or under different experimental conditions.

5. Multiplex IHC: In multiplex IHC, multiple antibodies are used simultaneously to detect and visualize multiple target antigens within the same tissue section. This allows for the analysis of multiple proteins or biomolecules within the same tissue sample. Multiplex IHC can be used to detect and visualize multiple proteins or biomolecules within the same tissue section, allowing for the analysis of complex signaling pathways and cellular interactions in tissues. For example, multiplex IHC can be used to visualize the interactions between immune cells and cancer cells in tumor tissues.

These different types of IHC can be combined with different detection methods, such as chromogenic or fluorescent detection, to provide a range of options for detecting and visualizing specific proteins or biomolecules within tissues.

---

PROTOCOL