**1. Introduction**

Immunohistochemistry is used for identifying the localization of cellular or tissue constituents (antigens) based on antigen-antibody interactions using labeled antibodies that can be visualized under light and electron microscopes. Therefore, the use of specific primary antibodies and tissue preparation techniques that conserve fine structures, the immobilization of antigens, and antigen-antibody interaction is essential. Until the end of the 1980s, the conservation of protein conformation was thought to be important for the immunohistochemistry of protein antigens for the following reasons. (1) The production and specificity of antibodies were mainly confirmed by immunoprecipitation using Ouchterlony double diffusion test and immunoelectrophoresis, in which native purified antigen proteins or tissue extracts were reacted with antisera to form precipitation lines at the proper antigen/antibody ratio in agar or agarose gels. (2) Strong immunoreactions were observed in tissues and cells that were fixed using formaldehyde within a short time or using other physical fixatives, such as cold acetone. (3) Frozen sections provided stronger immunostaining than paraffin sections, which denatured protein conformations during dehydration and embedding.

On the other hand, monoclonal antibodies began to be prepared in many laboratories in the 1980s, and commercially available monoclonal antibodies that recognized special cell types were being applied for pathological diagnosis. Western blotting and enzyme-linked immunosorbent assays (ELISAs) were introduced as the main techniques for detecting antigen-antibody reactions. Since these techniques involve the immobilization of both native and denatured antigens on membranes or plastic plates and antigen-antibody reactions are visualized using enzyme-labeled antibodies, these techniques have a higher sensitivity than immunoprecipitation methods independent of the antigen/antibody ratio. These new antibody preparation techniques and assay systems have gradually changed the concept that it is important to expose epitopes for immunoreactions rather than preserving the conformation of whole antigen molecules.

Histopathologists have made great efforts to use formalin-fixed and paraffinembedded (FFPE) specimens for immunohistochemistry, since such specimens have long been used as the standard for light microscopy and are archived in many biological and clinical laboratories. In the early 1970s, treatments with enzymes such as proteases, nucleases, and hyaluronidase and with protein denaturants were introduced to enable the partial recovery of immunostaining. The development of heat-induced antigen retrieval (HIAR), as reported by Shi et al. in 1991 for FFPE specimens, completely changed the concept of immunohistochemistry [1]. Although the mechanism of HIAR was originally a mystery, several studies have elucidated that heating cleaves chemical crosslinks (methylene bridges) formed by formaldehyde and exposes epitopes in tissues [2, 3]. HIAR is now applied not only to FFPE sections but also to frozen sections, cultured cells, physically fixed materials, and plastic embedded specimens for both light and electron microscopy [4]. It is also applied to other histochemical fields, such as in situ hybridization and lectin histochemistry. Furthermore, FFPE specimens are recognized as useful resources to study protein expression, DNA aberrations, and RNA expression in normal and diseased tissues [5, 6].

In this chapter, the following topics will be described, focusing on a flexible reconsideration of the concept of immunohistochemistry: (1) antigen-antibody interactions in tissues, (2) mechanisms of chemical fixation, (3) mechanisms of HIAR in formaldehyde-fixed specimens including exposure of highly masked epitopes, (4) HIAR in immunoelectron microscopy including the use of conventionally processed specimens embedded in epoxy resins, and (5) effects of antibody diluents on immunohistochemistry.
