Under anesthesia, IVCT was performed on the left eyeballs with natural light coming through windows, by pouring liquid isopentane -propane cryogen (−193 °C) cooled in liquid nitrogen. Morphology for the eyeball tissues with IVCT-FS was evaluated by HE staining and differential interference contrast images (Fig. 36.1a), and the rod and cone photoreceptors were observed as well-preserved areas without obvious ice crystal s at the light microscopic level.

The Glu-IR was obtained in thin sections of the eyeball specimens with IVCT followed by FS. The Glu was immunolocalized in the IS, outer and inner plexiform layers, and ganglion cell layer (Fig. 36.1b). The level of Glu has been reported to be highly detected in a subpopulation of amacrine cells and ganglion cells [20, 21]. However, the Glu was dispersively immunolocalized in the inner and outer plexiform layers and ganglion cell layer in the tissues with IVCT-FS. This was probably due to the distance from the initial frozen surface causing larger ice crystal formation . When photoreceptor cell layers were obliquely sectioned, Glu-IR was observed as tiny dot patterns, indicating that the Glu immunostaining was inside the photoreceptor cells. From these results, Glu was assumed to localize in the IS.

Without glutaraldehyde , Glu-IR was not obtained (Fig. 36.1f). To examine whether bovine serum albumin (BSA) is effective as a carrier or blocking protein for the following primary antibody reaction, sections were treated with fish gelatin, which was expected to effectively inhibit nonspecific binding of the antibodies , before the primary anti-Glu antibody incubation. In sections of the specimen prepared with IVCT followed by FS without chemical fixative s , Glu-IR was not obtained with the fish gelatin pretreatment. In contrast, the Glu-IR was clearly detected with the fish gelatin pretreatment in sections of the specimen prepared with IVCT followed by FS with glutaraldehyde. Thus, BSA is probably effective as a blocking reagent, and it became clear that the paraffin sections of the retinal specimens with IVCT-FS retained many Glu molecules, which can be more clearly immunostained by the anti-Glu antibody.

It is difficult to confirm how much Glu in tissues was lost during the FS step with chemical fixative s such as paraformaldehyde and glutaraldehyde . We assume that the immunolocalization with IVCT-FS reflects the original distribution . If the diffusion artifact of Glu had strongly appeared, Glu immunolocalization would have been distributed in much wider areas, such as the outer segment of photoreceptors . The intensity of the IR was changed with different concentrations of glutaraldehyde. This may be due to alteration of cross-linked tissue structures hindering some Glu reactivity against BSA and/or antibodies in the paraffin sections.

With IVCT-FS, the IS of photoreceptors was clearly immunopositive for Glu. Glu-IR obtained in some restricted areas in retinal tissue samples with IVCT probably reflects the normal in vivo metabolic conditions of the IS without anoxia or ischemia .