Methods in immunochemistry

Laboratory techniques using the foundations of immunology have been available for many years and the introduction to and use of immunochemistry in neuroscience, as a tool to localize and identify proteins, has revolutionized clinical diagnostic and basic science practices. Immunohistochemistry uses antibodies to map the nervous system and to pinpoint antigens to specific locations and subpopulations of cells in the brain. Western blotting is a powerful tool based on protein separation and is routinely used for the detection and characterization of specific proteins. In this case, the detection process that selectively identifies the proteins of interest also relies on the specificity and the affinity of antibodies for an antigen. Enzyme-linked immunosorbent assays (ELISA) also use antibodies or antigens, coupled to an easily assayed enzyme, to provide a quantitative concentration measurement of the antigen or antibody of interest. The commonality of all three techniques is the crucial antibody-antigen reaction. This chapter discusses the intricacies of this immunoreaction and focuses on the use of immunohistochemistry, Western blotting and enzyme-linked immunosorbent assays in basic science. All three of these common laboratory practices are discussed in great detail with specific focus on general principles, set-up, reagents, procedure, detection, visualization, quantification, quality control, and troubleshooting.