**2. Antigen-antibody reactions**

Antibodies recognize epitopes, which are small areas of antigen proteins but not the entire antigen molecule. When purified proteins from tissues or recombinant proteins are injected into animals, polyclonal antibodies that react with multiple epitopes and have different affinities and immunoglobulin classes for each epitope are generated. If a synthesized linear peptide is used for the immunogens, generated antibodies with different affinities and classes and subclasses of immunoglobulins may react with a single epitope. Therefore, we should notice that a commercially available polyclonal antibody may show different immunoreactions when we use the antibody with different lot numbers. On the other hand, a monoclonal antibody reacts with a single epitope with a monovalent affinity and a single immunoglobulin class and subclass. Antibodies prepared using the phage display technique have almost the same characteristics as monoclonal antibodies.

There are two types of epitopes, i.e., linear and conformational epitopes [4]. Linear epitopes are composed of a particular stretch of 5–20 consecutive amino

**31**

*Antigen Retrieval for Light and Electron Microscopy DOI: http://dx.doi.org/10.5772/intechopen.80837*

the selection of suitable diluents for antibodies.

**3. Mechanisms of chemical fixation**

same as those for 4% formaldehyde.

**3.1 Formaldehyde**

acids. Linear epitopes located on the surfaces of protein molecules can react with antibodies independent of whether the protein is in its native or denatured and extended states. Meanwhile, linear epitopes located on surfaces in contact with other proteins or the subunits and internal linear epitopes cannot bind to antibodies when the protein is in its native state and can only bind to antibodies when the polypeptides have been extended by heating or treatment with protein denaturants, such as sodium dodecyl sulfate (SDS) and urea. A few linear internal epitopes are rarely in contact with antibodies because the antigen proteins contain multiple disulfide bonds and the protein remains stable even after heating. The reduction of these disulfide bonds may be necessary for the exposure of such epitopes, as described later. Conformational epitopes are composed of amino acids that are located far apart in their linear sequence but become juxtaposed when the protein is folded into its native shape. Some conformational epitopes are stabilized by disulfide bonds and resistant to denaturation by heating and protein denaturants, but others associated with noncovalent forces may be sensitive to denaturation.

In immunohistochemistry, fixation is essential to conserve tissue structures and to prevent the diffusion of antigens. Chemical fixatives directly modify epitopes and crosslink proteins and nucleic acids to form gel-like structures that inhibit antigen-antibody interactions. The antigen-antibody reactions can be interfered with in special regions, such as cell organelles with intact membrane and secretory granules and deposits of protein fibrils that contain highly concentrated proteins, as well as in nuclei and extracellular matrices with highly negative electrostatic charges. Therefore, immunohistochemistry must be performed under conditions that promote the epitope-antibody association in the target tissues through the use of tissue-processing procedures, i.e., fixation, embedding, antigen retrieval, and

Chemical fixatives used for immunohistochemistry are limited to formaldehyde and glutaraldehyde. Formaldehyde is used for tissue fixation in both light and electron microscopy, while glutaraldehyde is used as a fixative only in electron microscopy. Although formaldehyde and glutaraldehyde are popular fixatives for histology and pathology, the characteristics and fixation mechanisms are assumed to be quite different. Since formalin is composed of about 35% formaldehyde aqueous solution containing about 10% methanol to prevent the polymerization of formaldehyde and is usually diluted 10-fold as 10% formalin, its fixation mechanisms should be the

The mechanism of fixation using formaldehyde is thought to be as follows. Formaldehyde forms an adduction of hydroxymethyl/methylol (CH2OH) to functional groups of amino acids (such as lysine, arginine, and cysteine) (**Figure 1a, d**, and **f**), the N-terminus of polypeptides, and bases of RNA and single strand DNA [7, 8]. A part of the methylol group of lysine and arginine forms imines (Schiff base) through the removal of H2O (**Figure 1a** and **d**), and the imines of lysine then combine with the side chains of amino acids, such as tyrosine (**Figure 1b**), tryptophan (**Figure 1c**), asparagine, glutamine, and histidine, and the imine of arginine (**Figure 1e**) to form approximately 0.25-nm methylene bridges (-CH2-). Although lysines are reported to be major reactive residues for formaldehyde in native proteins, only lysines located on the surface area are modified by formaldehyde [9]. Methylols of cysteine form methylene

#### *Antigen Retrieval for Light and Electron Microscopy DOI: http://dx.doi.org/10.5772/intechopen.80837*

*Immunohistochemistry - The Ageless Biotechnology*

preserving the conformation of whole antigen molecules.

RNA expression in normal and diseased tissues [5, 6].

almost the same characteristics as monoclonal antibodies.

on immunohistochemistry.

**2. Antigen-antibody reactions**

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

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

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

Antibodies recognize epitopes, which are small areas of antigen proteins but not the entire antigen molecule. When purified proteins from tissues or recombinant proteins are injected into animals, polyclonal antibodies that react with multiple epitopes and have different affinities and immunoglobulin classes for each epitope are generated. If a synthesized linear peptide is used for the immunogens, generated antibodies with different affinities and classes and subclasses of immunoglobulins may react with a single epitope. Therefore, we should notice that a commercially available polyclonal antibody may show different immunoreactions when we use the antibody with different lot numbers. On the other hand, a monoclonal antibody reacts with a single epitope with a monovalent affinity and a single immunoglobulin class and subclass. Antibodies prepared using the phage display technique have

There are two types of epitopes, i.e., linear and conformational epitopes [4]. Linear epitopes are composed of a particular stretch of 5–20 consecutive amino

**30**

acids. Linear epitopes located on the surfaces of protein molecules can react with antibodies independent of whether the protein is in its native or denatured and extended states. Meanwhile, linear epitopes located on surfaces in contact with other proteins or the subunits and internal linear epitopes cannot bind to antibodies when the protein is in its native state and can only bind to antibodies when the polypeptides have been extended by heating or treatment with protein denaturants, such as sodium dodecyl sulfate (SDS) and urea. A few linear internal epitopes are rarely in contact with antibodies because the antigen proteins contain multiple disulfide bonds and the protein remains stable even after heating. The reduction of these disulfide bonds may be necessary for the exposure of such epitopes, as described later. Conformational epitopes are composed of amino acids that are located far apart in their linear sequence but become juxtaposed when the protein is folded into its native shape. Some conformational epitopes are stabilized by disulfide bonds and resistant to denaturation by heating and protein denaturants, but others associated with noncovalent forces may be sensitive to denaturation.

In immunohistochemistry, fixation is essential to conserve tissue structures and to prevent the diffusion of antigens. Chemical fixatives directly modify epitopes and crosslink proteins and nucleic acids to form gel-like structures that inhibit antigen-antibody interactions. The antigen-antibody reactions can be interfered with in special regions, such as cell organelles with intact membrane and secretory granules and deposits of protein fibrils that contain highly concentrated proteins, as well as in nuclei and extracellular matrices with highly negative electrostatic charges. Therefore, immunohistochemistry must be performed under conditions that promote the epitope-antibody association in the target tissues through the use of tissue-processing procedures, i.e., fixation, embedding, antigen retrieval, and the selection of suitable diluents for antibodies.
