*5.4.2 HIAR in epon-embedded materials*

Partial deresination with sodium ethoxide was required for light microscopy using semi-thin epon sections. After autoclaving in 100 m Tris-HCl (pH 9.0) for 10 min, six of the seven antibodies that showed positive immunoreactions in the frozen sections exhibited clear localizations in the semi-thin sections. α-Amylase (**Figure 8A** and **B**), clathrin (**Figure 8C**), and claudin-5 (**Figure 8D**), which all showed positive immunoreactions in the semi-thin sections, were localized on ultrathin sections using colloidal gold-labeled antibodies. For HIAR, ultrathin sections were heated in 500 mM Tris-HCl buffer (pH 9.0) for 1–3 h at 95°C. Although heat treatment was essential for the detection of antigens on ultrathin sections, heat treatment reduced the electron density of ribosomes, chromatins,


*Fresh frozen sections were fixed with 2% glutaraldehyde/0.1 M phosphate buffer (pH 7.4) for 30 min (GA-EtOH) or fixed with 2% glutaraldehyde/0.1 M phosphate buffer (pH 7.4) for 30 min and further fixed with 1% osmium tetroxide/0.1 M phosphate buffer (pH 7.4) for 30 min (GA-OsO4-EtOH). Some sections fixed with glutaraldehyde and osmium tetroxide were further treated with 1% sodium metaperiodate aqueous solution for 5 min (NaIO4): all sections were hydrated with ethanol and then rehydrated after fixation. The fixed sections were immunostained after autoclaving in 20 mM Tris-HCl (pH 9.0) at 120°C for 10 min (autoclave) or without heat treatment (Non). Immunostaining was scored as followed: 3, strong; 2, moderate; 1, weak; 0–1, faint; and 0, negative.*

#### **Table 1.**

*HIAR in frozen sections prepared from specimens fixed with glutaraldehyde or glutaraldehyde and osmium tetroxide.*

#### **Figure 7.**

*HIAR in frozen sections fixed with glutaraldehyde and osmium tetroxide. Fresh frozen sections (6 μm) from mouse tissues were fixed with 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 min at room temperature (A, C, and E) and successively with 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) for 30 min at room temperature (B, D, and F). The sections were autoclaved in 20 mM Tris-HCl (pH 9.0) for 10 min at 120°C. They were then immunostained with anti-α-amylase antibody in the pancreas (A and B), anti-claudin-5 antibody in the kidney (C and D), and anti-clathrin antibody in the kidney (E and F). Although negative or weak immunostaining is seen in the sections fixed with glutaraldehyde after autoclave (A, C, and E), strong α-amylase immunoreactions are recognized in the apical cytoplasm of pancreatic acinar cells (B) and clear claudin-5 and clathrin immunoreactions are observed in the glomeruli (D) and in the apical cytoplasm of proximal tubular cells in the kidney (F), respectively. Bar = 50 μm.*

#### **Figure 8.**

*HIAR for the specimens fixed with glutaraldehyde and osmium tetroxide, embedded in epoxy resin. Mouse tissues were fixed with 2% glutaraldehyde/0.1 M phosphate buffer (pH 7.4) for 3 h at 4°C and post-fixed with 1% osmium tetroxide/0.1 M phosphate buffer (pH 7.4) for 1 h and then embedded in the epoxy resin. Ultrathin sections were heated in 0.5 M Tris-HCl (pH 9.0) for 2 h at 95°C. α-Amylase is localized in the Golgi apparatus (G), condensing vacuole and secretory granules in the exocrine pancreas after heat treatment (B), whereas no reaction is seen in the sections without heating (A). Immunoreaction for clathrin is recognized in the apical canaliculi of renal proximal tubular cell (C). Claudin-5 is localized along the membrane of podocyte foot processes in the glomerulus (D). Bar = 500 nm.*

**45**

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

microscopy are described.

**6.1 Kind of dilution buffers**

**6.2 Ionic strength of dilution solution**

localization compared with paraffin-embedded materials.

**6. Effects of diluents on antibodies in immunohistochemistry**

The relationship between epitopes and paratopes of antibodies is thought to be similar to that between keys and keyholes. However, since these structures change their conformations to form a final specific and tight binding after antigen-antibody association, conservation of the flexibility of their polypeptide chains should be important. Although hydrogen bonds, hydrophobic forces, electrostatic forces, and van der Waals forces all participate in the final tight binding, electrostatic forces are important for the initial contact and association of antigen and antibody molecules (i.e., the net charges of each molecule and the neighboring charges of antigens). Buffer type, ionic strength, pH, and the presence of detergents in solutions are likely to exert strong influences on the antigen-antibody reaction. Although many kinds of diluents are commercially available for immunohistochemistry and Western blotting and yield good results with low background staining and a high sensitivity for some antigens, systematic studies of antibody diluents for immunohistochemistry have not been performed. In this section, the effects of dilution solutions for primary antibodies on immunostaining for light and electron

Fifteen monoclonal antibodies were diluted in 10 mM phosphate buffer (pH 7.4)

Fifteen monoclonal antibodies were diluted in 1% BSA/10 mM Tris-HCl buffer (pH 7.4) containing 50 mM NaCl, 150 mM NaCl, or 300 mM NaCl. After autoclaving, the paraffin sections were treated with the primary antibodies overnight and then with Envision HRR for 1 h at room temperature. The results are shown in **Table 2**. Most of the antibodies showed strong immunostaining when they were diluted with a buffer containing 50 mM NaCl [45]. However, monoclonal antibodies to proliferating cell nuclear antigen (PCNA) showed the strongest immunostaining when diluted with a buffer containing 300 mM NaCl, and monoclonal antibodies to glial fibrillary

containing 150 mM NaCl (PBS), 10 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl (TBS), or 10 mM FEPES-NaOH buffer (pH 7.4) containing 150 mM NaCl (HBS); 1% bovine serum albumin (BSA) (final concentration) was added to each solution. Paraffin sections from mouse tissues fixed with formaldehyde were immunostained after autoclaving in 20 mM Tris-HCl buffer (pH 9.0) for 10 min. The sections were treated with the primary antibodies diluted with the solutions overnight at 4°C and successively with Envision HRR (Dakocytomation) for 1 h at room temperature. As shown in **Table 2**, all the antibodies diluted with TBS showed stronger immunoreactions than those diluted with PBS or HBS [45]. Although the reasons are unclear, the binding of phosphate ion (a larger ion) to positively charged regions of epitopes and paratopes may reduce the flexibility of peptide chains.

intracellular membranes, and secretory granules in the exocrine pancreas. The partial removal of epoxy resins with sodium ethoxide followed by autoclaving revealed the disruption of the fine structure and no reproducible immunolabeling. These results indicated that archived epon-embedded specimens could be a useful resource for immunohistochemical studies at both the light and electron microscopy levels, since they provide excellent morphology and detailed antigen

*Immunohistochemistry - The Ageless Biotechnology*

**44**

**Figure 8.**

**Figure 7.**

*processes in the glomerulus (D). Bar = 500 nm.*

*HIAR for the specimens fixed with glutaraldehyde and osmium tetroxide, embedded in epoxy resin. Mouse tissues were fixed with 2% glutaraldehyde/0.1 M phosphate buffer (pH 7.4) for 3 h at 4°C and post-fixed with 1% osmium tetroxide/0.1 M phosphate buffer (pH 7.4) for 1 h and then embedded in the epoxy resin. Ultrathin sections were heated in 0.5 M Tris-HCl (pH 9.0) for 2 h at 95°C. α-Amylase is localized in the Golgi apparatus (G), condensing vacuole and secretory granules in the exocrine pancreas after heat treatment (B), whereas no reaction is seen in the sections without heating (A). Immunoreaction for clathrin is recognized in the apical canaliculi of renal proximal tubular cell (C). Claudin-5 is localized along the membrane of podocyte foot* 

*HIAR in frozen sections fixed with glutaraldehyde and osmium tetroxide. Fresh frozen sections (6 μm) from mouse tissues were fixed with 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 min at room temperature (A, C, and E) and successively with 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) for 30 min at room temperature (B, D, and F). The sections were autoclaved in 20 mM Tris-HCl (pH 9.0) for 10 min at 120°C. They were then immunostained with anti-α-amylase antibody in the pancreas (A and B), anti-claudin-5 antibody in the kidney (C and D), and anti-clathrin antibody in the kidney (E and F). Although negative or weak immunostaining is seen in the sections fixed with glutaraldehyde after autoclave (A, C, and E), strong α-amylase immunoreactions are recognized in the apical cytoplasm of pancreatic acinar cells (B) and clear claudin-5 and clathrin immunoreactions are observed in the glomeruli (D) and in the apical* 

*cytoplasm of proximal tubular cells in the kidney (F), respectively. Bar = 50 μm.*

intracellular membranes, and secretory granules in the exocrine pancreas. The partial removal of epoxy resins with sodium ethoxide followed by autoclaving revealed the disruption of the fine structure and no reproducible immunolabeling.

These results indicated that archived epon-embedded specimens could be a useful resource for immunohistochemical studies at both the light and electron microscopy levels, since they provide excellent morphology and detailed antigen localization compared with paraffin-embedded materials.
