Immunohistochemical Protocols

Abstract

In this issue of the Journal, Shi et al 1 shatter yet another myth of tissue preparation for immunohistochemical studies, and, in doing so, they send us back to the future with antibody protocol development for immunohistochemistry in general. The authors examined 26 antibodies with the avidinbiotin method and found that more than half of the tested antibodies showed immunohistochemical staining results that were indistinguishable between fresh acetone-fixed and neutral buffered formalin (NBF)-fixed tissue sections. An additional third of the tested antibodies showed better immunohistochemical signals following NBF and antigen retrieval (AR); only 2 antibodies gave better immunohistochemical staining results for acetone-fixed tissue sections. It is important to note that cytoplasmic proteins showed comparable immunohistochemical signals between acetone- and NBF-fixed tissue sections. Nuclear proteins fixed in NBF also gave better immunohistochemical signals than those obtained by fresh tissue fixed in acetone. Comparable results were obtained by Yamashita and Okada 2 when comparing an aldehyde fixative with acetone on fresh tissue. This new work from this pioneering group comes as no surprise because they were also instrumental in revolutionizing the practice of immunohistochemistry with the development of AR. AR has leveled the playing field of immunohistochemistry and was a huge step in the direction of standardization of methods for diagnostic immunohistochemistry for the discipline of surgical pathology. The bold step that resulted in development of AR challenged the traditional view that tissue exposure to high temperatures was detrimental to tissue antigens. The view that less heat is better for the tissue sections was thrown aside when Shi et al 3 found that heating tissue sections in liquids at high temperatures vastly improved immunostaining for a large number of antibodies. AR is now a necessary step in the evaluation of new antibodies introduced into the clinical immunohistochemistry laboratory. AR is an integral component of the workup to optimize a new antibody in the laboratory. AR may be performed in microwaves, pressure cookers, or online in automated instruments, 4 and it also involves the use of buffers