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, laboratory oven, water bath, pressure cooker, autoclave, or a vegetable steamer. This technique was developed by Dr. Shan-Rong Shi and Dr. Clive Taylor, who discovered that the degree of epitope recovery is dependent upon a number of factors, including the timing of when heating occurs, as well as the temperatures involved.
The pH of the retrieval solution is also a very important factor. A pH that is too high or too low will alter the final staining results. For example, too high heat or too high pH could cause detachment or washing of the tissue off the slide. There is no 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. Much like dilutions for concentrated primary antibodies, antibody manufacturers make recommendations for the appropriate HIER solution that should be used. However, the effectiveness of the HIER solution and method should be assessed by individual laboratories based on length and method of tissue fixation.
