**4. Mechanisms of HIAR**

After the first report for HIAR by Shi et al. [1], investigators have tried to select the most suitable heating conditions (heating devices, temperatures, kinds of solutions, solution pH, and additives). However, the total amount of applied heat energy is now recognized as being more important than the type of heating devices. In this section, the effects of pH and the ionic strength of retrieval solutions for HIAR will be reviewed, and the mechanisms of HIAR will be described.

### **4.1 Effects of pH on proteins treated with formaldehyde**

When purified proteins are treated with formaldehyde and analyzed using SDS-PAGE (polyacrylamide gel electrophoresis), protein oligomers formed by intermolecular crosslinks were recognized. Monomer and oligomers treated with formaldehyde showed smaller apparent molecular weight compared with those of unmodified native proteins, since intramolecular crosslinks prevented the complete unfolding of proteins in the SDS solution [2, 18, 19]. The cleaving efficiency of the crosslinks was almost the same when the formaldehyde-treated proteins were heated for 5 min at 100°C in 10 mM Tris-HCl at pH 3.0, pH 6.0, pH 7.5, or pH 9.0 while analyzed with SDS-PAGE. When the proteins were drastically heated by autoclaving for 10 min at 120°C at a pH close to their respective isoelectric points, the proteins tended to produce insoluble protein precipitates [2]. However, many investigators have demonstrated that the efficiency of HIAR for immunohistochemistry is highly dependent on the pH of the retrieval solution.

#### **4.2 Effect of pH of HIAR solutions on immunohistochemistry**

Shi et al. systematically studied the effects of the pH of antigen retrieval solution on HIAR [20]. They classified the pH-influenced HIAR immunostaining patterns as follows: type A, in which staining was almost the same at any pH, with a slight decrease in intensity between pH 3.0 and pH 6.0; type B, in which a dramatic increase in immunostaining was observed at acidic and basic pH; and type C, in which the immunostaining intensity increased at basic pH. We re-examined the pH dependency of HIAR using 17 different antibodies and observed two immunostaining patterns for the pH dependency of HIAR [3]. The majority of the antibodies produced the first immunostaining pattern; that is, they yielded a positive

**35**

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

epitopes composed of basic amino acids [23].

**4.4 Effect of ionic strength of HIAR solution**

**4.5 Mechanisms of HIAR in FFPE sections**

investigate temporal interactions [8, 25, 26].

epitopes in HIAR.

**4.3 pH-dependent reversibility of HIAR efficiency**

immunoreaction when heated in buffers that had either an acidic pH or a basic pH. This HIAR pattern may correspond to the type-B pattern of the classification by Shi et al. If highly diluted antibodies had been used in the immunohistochemical studies, the type-A pattern described by Shi et al. might have become nearly equivalent to the type**-**B pattern. The second immunostaining pattern that we observed was a strong immunostaining reaction when heated in basic buffer, corresponding to the type-C pattern described by Shi et al. Pileri et al. and Kim et al. have also reported that a basic buffer is effective for HIAR for most antigens [21, 22]. On the other hand, Kajiya et al. reported that heating at an acidic pH (pH 3.0 or pH 6.0) frequently enabled excellent immunostaining for the detection of basic proteins or

Yamashita and Okada demonstrated that the intensities of the immunoreactions obtained by heating in a buffer are reversibly altered by successive heating in another buffer with a different pH [2]. For example, when the first heating in a buffer (pH 6.0) yielded a weak immunostaining in FFEP sections, a second heating at pH 9.0 significantly increased the immunostaining; however, the third heating in the acidic buffer weakened the immunostaining. These results indicate that the degradation or extraction of antigens is not a major factor in the pH dependency of HIAR and that the pH of the solution is a critical factor for the exposure of tissue

We studied the effects of ionic strength on HIAR using 10 antibodies. Three buffer systems with different pH values were examined. When FFPE specimens were autoclaved for 10 min at 120°C in 20 mM Tris-HCl buffer (pH 9.0), 50 mM citraconic anhydride aqueous solution (pH 7.4), or 10 mM citrate buffer (pH 6.0) containing 0, 50, 100, or 200 mM NaCl, all the antibodies showed the strongest immunostaining while the sections were autoclaved in the NaCl-free solutions. The staining intensity decreased as the NaCl concentration increased in all antibodies examined [24]. These results demonstrated that the ionic strength of the solution is a critical factor for HIAR and that a high concentration of salt inhibits the exposure of epitopes.

The results described above demonstrate that the fundamental mechanism of HIAR is based on the cleavage of protein-protein crosslinks and the exposure of epitopes. Heating destroys the gel-like structure formed by formaldehyde-fixation and partially extracts the macromolecules, enabling the antibodies to penetrate tissues easily; this process is similar to the effects of enzyme digestion. Western blot analyses have demonstrated that soluble, nuclear, and membrane proteins are extracted from FFPE specimens after heating but not from those without heat treatment [2]. Recent proteomics studies using a mass spectrum technique have also revealed that heating facilitates protein extraction from archived FFPE specimens [6]. Furthermore, heatinduced cleaving of the shortest crosslinks induced by formaldehyde can be applied to chromatin immunoprecipitation assays and to the crosslinks of adjacent proteins to

The second mechanism is assumed to be as follows based on the pH-dependent

and ionic strength-dependent phenomena described above [3, 4]. When the methylene bridges are cleaved by heating, the higher order structure of the protein is destroyed and the polypeptide chains are extended, exposing both hydrophobic

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

*Immunohistochemistry - The Ageless Biotechnology*

**3.3 Osmium tetroxide**

**4. Mechanisms of HIAR**

Since the cross-linked proteins rapidly form harder gel-like structures, compared with those created by formaldehyde, only thin layers of tissues can be fixed well using immersion fixation. Since aldehyde residues remain in the tissues fixed with glutaraldehyde, the aldehyde should be quenched using amides, such as glycine, ammonium chloride, and tris(hydroxymethyl) aminomethane, or reduced

Since osmium tetroxide binds to the unsaturated bonds of fatty acids and fixes membrane lipids, providing contrast by scattering electron beams, it is used as the post-fixing reagent after glutaraldehyde fixation in electron microscopy. Osmium tetroxide should also bind to the carbon-carbon double bonds formed by glutaraldehyde fixation (**Figure 2c**). However, since osmium tetroxide cleaves polypeptides in tryptophan residues and oxidizes methionine to methionine sulfone and cysteine to cysteic acid [17], osmium tetroxide significantly inhibits immunoreactions.

After the first report for HIAR by Shi et al. [1], investigators have tried to select

the most suitable heating conditions (heating devices, temperatures, kinds of solutions, solution pH, and additives). However, the total amount of applied heat energy is now recognized as being more important than the type of heating devices. In this section, the effects of pH and the ionic strength of retrieval solutions for

When purified proteins are treated with formaldehyde and analyzed using SDS-PAGE (polyacrylamide gel electrophoresis), protein oligomers formed by intermolecular crosslinks were recognized. Monomer and oligomers treated with formaldehyde showed smaller apparent molecular weight compared with those of unmodified native proteins, since intramolecular crosslinks prevented the complete unfolding of proteins in the SDS solution [2, 18, 19]. The cleaving efficiency of the crosslinks was almost the same when the formaldehyde-treated proteins were heated for 5 min at 100°C in 10 mM Tris-HCl at pH 3.0, pH 6.0, pH 7.5, or pH 9.0 while analyzed with SDS-PAGE. When the proteins were drastically heated by autoclaving for 10 min at 120°C at a pH close to their respective isoelectric points, the proteins tended to produce insoluble protein precipitates [2]. However, many investigators have demonstrated that the efficiency of HIAR for immunohistochem-

Shi et al. systematically studied the effects of the pH of antigen retrieval solution on HIAR [20]. They classified the pH-influenced HIAR immunostaining patterns as follows: type A, in which staining was almost the same at any pH, with a slight decrease in intensity between pH 3.0 and pH 6.0; type B, in which a dramatic increase in immunostaining was observed at acidic and basic pH; and type C, in which the immunostaining intensity increased at basic pH. We re-examined the pH dependency of HIAR using 17 different antibodies and observed two immunostaining patterns for the pH dependency of HIAR [3]. The majority of the antibodies produced the first immunostaining pattern; that is, they yielded a positive

HIAR will be reviewed, and the mechanisms of HIAR will be described.

**4.1 Effects of pH on proteins treated with formaldehyde**

istry is highly dependent on the pH of the retrieval solution.

**4.2 Effect of pH of HIAR solutions on immunohistochemistry**

to alcohols using sodium borohydride prior to immunostaining.

**34**

immunoreaction when heated in buffers that had either an acidic pH or a basic pH. This HIAR pattern may correspond to the type-B pattern of the classification by Shi et al. If highly diluted antibodies had been used in the immunohistochemical studies, the type-A pattern described by Shi et al. might have become nearly equivalent to the type**-**B pattern. The second immunostaining pattern that we observed was a strong immunostaining reaction when heated in basic buffer, corresponding to the type-C pattern described by Shi et al. Pileri et al. and Kim et al. have also reported that a basic buffer is effective for HIAR for most antigens [21, 22]. On the other hand, Kajiya et al. reported that heating at an acidic pH (pH 3.0 or pH 6.0) frequently enabled excellent immunostaining for the detection of basic proteins or epitopes composed of basic amino acids [23].

### **4.3 pH-dependent reversibility of HIAR efficiency**

Yamashita and Okada demonstrated that the intensities of the immunoreactions obtained by heating in a buffer are reversibly altered by successive heating in another buffer with a different pH [2]. For example, when the first heating in a buffer (pH 6.0) yielded a weak immunostaining in FFEP sections, a second heating at pH 9.0 significantly increased the immunostaining; however, the third heating in the acidic buffer weakened the immunostaining. These results indicate that the degradation or extraction of antigens is not a major factor in the pH dependency of HIAR and that the pH of the solution is a critical factor for the exposure of tissue epitopes in HIAR.

### **4.4 Effect of ionic strength of HIAR solution**

We studied the effects of ionic strength on HIAR using 10 antibodies. Three buffer systems with different pH values were examined. When FFPE specimens were autoclaved for 10 min at 120°C in 20 mM Tris-HCl buffer (pH 9.0), 50 mM citraconic anhydride aqueous solution (pH 7.4), or 10 mM citrate buffer (pH 6.0) containing 0, 50, 100, or 200 mM NaCl, all the antibodies showed the strongest immunostaining while the sections were autoclaved in the NaCl-free solutions. The staining intensity decreased as the NaCl concentration increased in all antibodies examined [24]. These results demonstrated that the ionic strength of the solution is a critical factor for HIAR and that a high concentration of salt inhibits the exposure of epitopes.

#### **4.5 Mechanisms of HIAR in FFPE sections**

The results described above demonstrate that the fundamental mechanism of HIAR is based on the cleavage of protein-protein crosslinks and the exposure of epitopes. Heating destroys the gel-like structure formed by formaldehyde-fixation and partially extracts the macromolecules, enabling the antibodies to penetrate tissues easily; this process is similar to the effects of enzyme digestion. Western blot analyses have demonstrated that soluble, nuclear, and membrane proteins are extracted from FFPE specimens after heating but not from those without heat treatment [2]. Recent proteomics studies using a mass spectrum technique have also revealed that heating facilitates protein extraction from archived FFPE specimens [6]. Furthermore, heatinduced cleaving of the shortest crosslinks induced by formaldehyde can be applied to chromatin immunoprecipitation assays and to the crosslinks of adjacent proteins to investigate temporal interactions [8, 25, 26].

The second mechanism is assumed to be as follows based on the pH-dependent and ionic strength-dependent phenomena described above [3, 4]. When the methylene bridges are cleaved by heating, the higher order structure of the protein is destroyed and the polypeptide chains are extended, exposing both hydrophobic

and hydrophilic regions and epitopes. The polypeptide chains then rapidly refold during the cooling process. In tissues, many kinds of proteins with different isoelectric points and molecular weights are tightly packed, and neighboring polypeptides can come in contact with each other. Therefore, epitopes should be concealed during the refolding of the proteins at around a neutral pH because a strong hydrophobic attractive force would randomly entangle the neighboring polypeptides: an electrostatic force may act locally as either an attractive or a repulsive force. At basic or acidic pH values, however, the majority of the extended polypeptides would be charged negatively or positively, and the electrostatic repulsive force would act to prevent random aggregation and entanglement with neighboring polypeptides caused by the hydrophobic force, thereby maintaining a suitable extend conformation for antigen-antibody interactions. When salt is added to the retrieval solutions, the electrostatic force between neighboring polypeptides is canceled, and the hydrophobic attractive force may cause the antigen proteins and neighboring proteins to aggregate, masking the epitopes. Namimatsu et al. reported that heating in citraconic anhydride solution at a neutral pH was useful as a universal antigen retrieval method [27]. Since citraconic anhydride binds to the ε-amino groups of lysine residues re-exposed after heating and places numerous negative charges on proteins, an electric repulsive force may help to keep polypeptides in an unfolded state.

On the other hand, strong heating at around the isoelectric points of proteins induces their coagulation [2], and increasing the ionic strength also promotes isoelectric precipitation. Therefore, many proteins with neutral isoelectric points can be precipitated in a solution with a neutral pH. The finding that an acidic buffer is effective for some basic antigens probably indicates that these antigens are precipitated by heating in basic buffers [23, 28]. Since heating may destroy the protein conformation, most conformational epitopes associated with noncovalent forces should lose their reactivity to antibodies. On the other hand, HIAR may be effective for conformational epitopes that have been stabilized by disulfide bonds.

Basic or acidic solutions are effective for HIAR as described above, whereas citrate buffer (pH 6.0) is frequently used in pathological studies. Citrate buffer may be suitable for examining detailed nuclear structures, since heating in basic solutions cleaves and extracts RNAs and reduces the nuclear stainability with hematoxylin. In practice, at least three antigen retrieval solutions at pH 3.0, pH 6.0, and pH 9.0 should be examined when studying the localization of unknown antigens for the first time.
