**5.2 Pre-embedding method**

*Immunohistochemistry - The Ageless Biotechnology*

**5. HIAR in immunoelectron microscopy**

ecules also severely inhibit antigen-antibody interactions.

antigen independent of the staining methods.

osmium tetroxide and embedded in epoxy resins.

**5.1 Standardized fixative**

fixatives followed by heating (**Figure 3B** and **D**). In such cases, the epitopes should be located in a highly folded portion of the antigen protein that may be stabilized with disulfide bonds to form stable secondary and tertiary structures. The reduction of disulfide bonds using dithiothreitol or 2-mercaptoethanol prior to heat treatment yields strong immunoreactions (**Figure 3C** and **E**) [28, 30]. In addition, when epitopes are covered by neighboring heat-resistant polypeptides, the accessibility of antibodies to the epitopes is also inhibited. When immunostaining is performed using old FFEP or using sections on a slide glass stored for a long time, the samples should be oxidized to produce many disulfide bonds in the tissues. The reduction and cleavage of disulfide bonds may be effective for such specimens (**Figure 3F–I**).

Immunoelectron microscopy is a powerful technique for observing the localization of antigens in cell organelles and for studying the relationship between antigens and other macromolecules. Three main immunoelectron microscopy methods have been used to localize antigens: the pre-embedding method, the post-embedding method, and cryoultramicrotomy. Although fixation is one of the most important aspects of sample preparation for all three methods, the choices of fixatives and tissue processing procedures are limited unlike light microscopy, because of the need to satisfy compatible requirements, such as conservation of fine structure, immobilization of antigen minimizing the diffusion, and conservation of immunoreaction. In general, fixatives that allow good morphological findings and precise antigen localization through the rapid and tight crosslinking of macromol-

Fixatives containing formaldehyde as the main crosslinking regent are popular for immunoelectron microscopy using the pre-embedding method, since antibody penetration into the cells is an important factor for this method; formaldehyde solution, PLP (periodate-lysine-paraformaldehyde) [31], and a mixture of formaldehyde and a low concentration (0.05–0.5%) of glutaraldehyde. For the post-embedding method, a mixture of formaldehyde and a low concentration of glutaraldehyde is frequently used to preserve the fine structure and membrane structures, since dehydration and resin embedding are performed without osmium tetroxide post-fixation. A short period of perfusion fixation with glutaraldehyde is also applied for the post-embedding method. The suitable fixatives, fixing periods, and temperatures of fixatives have been determined by trial and error for each

In this section, a standardized fixation method that yields positive immunoreactions for the pre-embedding and the post-embedding methods after HIAR will be described and discussed how HIAR is also effective for some routinely processed materials for the electron microscopy that are fixed with glutaraldehyde and

We introduced a standardized fixative that can yield positive immunoreactions for many antigens in electron microscopy after HIAR [32]. Tissues were fixed with 4% formaldehyde containing 2.5 mM CaCl2 and 1.25 mM MgCl2 in 0.1 M HEPES-NaOH buffer (pH 7.4) for 2 h at room temperature and then with the same fixative composition in 0.1 M HEPES buffer-NaOH buffer (pH 8.5) overnight at room temperature. The vehicle osmolarity of the fixatives was adjusted to 300–330 mOsm by adding sucrose or glucose. Formaldehyde containing CaCl2 and MgCl2 was shown to

**38**

Although the pre-embedding method is the most popular and the simplest method for immunoelectron microscopy, HIAR has only been applied for the detection of a few antigens. Frozen sections or vibratome sections from specimens fixed with formaldehyde or a mixture of formaldehyde and glutaraldehyde were heated in various solutions such as citrate buffer (pH 6.0), Tris-HCl buffer (pH 9.0 or pH 10.0), or citraconic anhydride solution (pH 7.4) for different periods for each antigen [33–36]. Yamashita reported that 4% formaldehyde containing 25 mM CaCl2 in 0.1 M cacodylate buffer (pH 7.4) was a suitable fixative for the pre-embedding method by applying HIAR for several antigens [4].

We applied the pre-embedding method to tissues fixed using the standardized fixative described above. Frozen sections (about 15 μm) were mounted on a slide glass and then heated in 20 mM Tris-HCl (pH 9.0) containing 10% sucrose for 2–4 h at 70°C. Immunostaining was performed using (horseradish peroxidase) HRP-labeled antibodies and antigen localization was visualized with 3,3′diaminobenzidine (DAB). Most of the antigens that were examined showed negative immunoreactions without heat treatment, but they produced strong immunoreaction after heating (**Figure 4**). Since endogenous immunoglobulins are inactivated after heat treatment (**Figure 4A** and **B**), the immunoreactions can be clearly detected even in the mouse tissues using mouse monoclonal antibodies. Tris-HCl buffer (pH 9.0) is effective for most antigens but citrate buffer (pH 6.0 or pH 3.0) yields strong reaction for a few antigens with basic isoelectric points, such as vascular endothelial cell growth factor (VEGF). Therefore, the selection of suitable solutions for each antigen should be examined using FFEP sections or frozen sections on light microscopy.

The positively immunostained sections were then post-fixed with 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) for 30 min, dehydrated with ethanol, and then embedded in epoxy resin. All antigens detected in frozen sections on light microscopy were localized using the pre-embedding method preserving fine structures (**Figure 5**).
