HIER involves treating the slides in heated solutions of various buffers and pH ranges that are used to break the cross-links before application of the primary antibody. Heat sources can be a microwave oven, oven, water bath, pressure cooker, autoclave, or a vegetable steamer. This technique was developed by Dr. Shan-Rong Shi, Taylor, and colleagues, who discovered that the degree of epitope recovery is a function of a number of factors, including the timing of when heating occurs and the temperatures involved.
The pH of the retrieval solution is also a very important factor to consider. A pH that is too high or too low will alter the final results. For example, too high heat or too high pH could cause detachment or washing of the tissue off the slide. There is not a universal HIER solution or heating method that is optimal for all antigens. One of the MOST common HIER solutions is 0.1 M citrate buffer (pH 6.0). Others used are 0.1 M EDTA (pH 8), 0.5 M Tris base buffer (pH 10), 0.05 M glycine-HCl buffer, and 1% periodic acid, to name a few. Each lab should investigate what method is BEST for the antibodies they use.