Immunoreactivity of Glutamate in Mouse Retina Inner Segment of Photoreceptors With In Vivo Cryotechnique

Dot-blot Analysis for Bovine Serum Albumin (BSA), BSA-conjugated Glu, and Glu Against the Anti-Glu Antibody

The antibody used for the immunohistochemistry in this study was a commercially available anti-Glu antibody (cat #G6642; Sigma-Aldrich Corp., St. Louis, MO), which was developed in rabbits using purified Glu conjugated to keyhole limpet hemocyanin (KLH) as the immunogen. According to the manufacturer's instructions, it reacts with Glu-KLH, Glu-BSA, KLH, and L-Glu, but not with BSA, using a dot-blot immunobinding assay. In this study, we reconfirmed the specific IR with dot-blot analysis, as described below. One microliter of BSA (10 mg/ml; Sigma) was placed on a nitrocellulose membrane (Millipore; Billerica, MA). BSA-dotted membranes were treated with 0.1% glutaraldehyde at room temperature for 1 hr, then some were treated with Glu (2 mg/ml; Sigma) at room temperature for 1 hr. All were treated with 5% fish gelatin (cat #G7765; Sigma) at room temperature for 2 hr. Then they were incubated with the same rabbit anti-Glu antibody that was used for immunohistochemistry (1:30,000 in dilution) at 4C overnight, followed by a biotinylated anti-rabbit IgG antibody (1:3000; Vector Laboratories, Inc., Burlingame, CA) at room temperature for 1 hr and a peroxidase-labeled avidin-biotin complex (ABC) (1:3000; Pierce, Rockford, IL) at room temperature for 30 min. The immunoreaction products were visualized with a metal-enhanced diaminobenzidine (DAB) reaction (Pierce). As a positive immunocontrol, the BSA dot on the nitrocellulose membrane was treated with fish gelatin, immunostained with a sheep anti-BSA antibody (Bethyl Laboratories; Montgomery, TX), and visualized in the same way as described above.

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Figure 1

A flow diagram of the preparation steps for the mouse eyeball tissues, as prepared by the in vivo cryotechnique (IVCT) (A) and freeze-substitution (FS) fixation for the glutamate (Glu) immunostaining. During the FS, paraformaldehyde, glutaraldehyde, or no fixative was added to acetone (B). Some thin sections of eyeball tissues without the chemical fixative during FS were treated with paraformaldehyde or glutaraldehyde (C). Before immunoreaction of the primary antibody, a common blocking treatment with bovine serum albumin (BSA) or fish gelatin was performed on the sections (D). PFA, paraformaldehyde; GA, glutaraldehyde; Ab, antibody; ABC-DAB, horseradish-avidin-biotin complex and diaminobenzidine reactions.

To evaluate the antibody IR against Glu alone, a dotblot analysis on glass slides was performed, as described previously (Terada et al. 2006). Briefly, ionized amine residue-coated glass slides (MAS glass slide; Matsunami Glass Ind. Ltd, Osaka, Japan) were treated with glutaraldehyde, and then Glu (Sigma) was additionally attached. The Glu-attached glass slides were subsequently treated with BSA, the primary anti-Glu antibody, and a secondary AlexaFluor 488–conjugated anti-rabbit IgG antibody (Invitrogen Corp., Carlsbad, CA). For the immunocontrol study, the primary antibody was omitted. The treated samples were observed in a fluorescence microscope (BX-61; Olympus, Tokyo, Japan).

IVCT for Living Mouse Eyeballs

Ten adult C57BL/6 mice weighing 20–30 g were used. Under pentobarbital anesthesia, IVCT was performed on the left eyeballs with natural light coming through windows (1355 ± 23 lx) (Figure 1A), by pouring 50 ml liquid isopentane-propane cryogen (-193C) cooled in liquid nitrogen. The low temperature was maintained by pouring extra liquid nitrogen on the eyeballs (Terada et al. 2006). The mice were always under normal physiological conditions, with their hearts beating (Ohno et al. 1996). The frozen eyeballs were obtained using a dental drill in liquid nitrogen, and processed for the next FS step.

FS Fixation With Various Chemical Fixatives for Paraffin Embedding

The frozen retinal tissues were processed for FS in acetone containing 2% paraformaldehyde, and 0.14%, 1.4%, or 14% glutaraldehyde, or no chemical fixative (Figure 1B), which was prepared using a molecular sieve (type 3A; Nakarai Tesque, Kyoto, Japan) for complete dehydration (Ohno et al. 2006). The frozen eyeballs were freeze-substituted at −80C for 24 hr and then put into a deep freezer at −30C and −10C for 2 hr each. Finally, they were placed in a refrigerator at 4C for 2 hr. They were warmed at room temperature for 1 hr, washed in pure acetone, immersed in xylene, and embedded in paraffin wax, as reported previously (Terada et al. 2007). The paraffin-embedded specimens were processed for the immunohistochemical step.

Immunostaining of Glu on Paraffin Sections

The paraffin-embedded eyeball tissues were cut at 5 μm thickness and routinely deparaffinized with xylene, a graded series of ethanol, and phosphatebuffered saline (PBS). Some thin sections were stained with hematoxylin-eosin (HE) for pure morphology. For other tissue sections without fixative during FS, 2% paraformaldehyde or 1.25% glutaraldehyde in 0.1 M PB was used for 1 hr at room temperature (Figure 1C). Some sections were treated by microwaving for 3 min in citrate buffer (pH 6.0). For immunostaining, they were pretreated with 5% BSA or 5% fish gelatin (Sigma) in PBS (Figure 1D), and with a rabbit polyclonal anti-Glu antibody at 1:10,000–30,000 dilutions (cat #G6642; Sigma) at 4C overnight. They were additionally treated with a biotinylated anti-rabbit IgG antibody (Vector) at room temperature for 1 hr, then a peroxidase-labeled ABC (Pierce) at room temperature for 1 hr, and visualized with metal-enhanced DAB (Pierce). Finally, they were incubated in 0.04% osmium tetroxide solution for 30–60 sec to increase and protect the contrast of imunoreaction products, and observed under a light microscope.

Immunolocalization of Glu in Retinas Reflecting Their Living State

First, to evaluate the IR against Glu-BSA, BSA or Glu alone of the commercially available anti-Glu antibody (cat #G6642; Sigma) dot-blot analysis was performed (Figure 2). The antibody reacted to the Glu-BSA (Figure 2A); however, it did not react to BSA itself (Figures 2C and 2E). In addition, IR was also obtained against Glu alone attached to glass slides with glutaraldehyde (Figure 2H), but it was not detected without the primary antibody (Figure 2I). These findings confirmed that the antibody reacts to Glu-BSA or Glu alone, but not to BSA itself.

Morphology for the eyeball tissues with IVCT-FS was evaluated by HE staining, and the rod and cone photoreceptors were observed as well-preserved areas without obvious ice crystals at the light microscopic level (Figure 3A). The Glu-IR was obtained in thin sections of the eyeball specimens with IVCT followed by FS with paraformaldehyde (Figure 3B). The Glu was immunolocalized in the IS, outer and inner plexiform layers, and ganglion cell layer, judged by HE staining of an adjacent section. Although the level of Glu has been reported to be highly detected in a subpopulation of amacrine cells and ganglion cells (Sun et al. 2007a, b), 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. At a higher-magnified view of the sections, in which photoreceptor cell layers were obliquely sectioned (Figures 3D and 3E), Glu-IR was observed as tiny dot patterns (arrows in Figure 3E), probably indicating that the Glu immunostaining was inside the photoreceptor cells. From these results, Glu was assumed to localize in the IS.

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Figure 2

Dot-blot analysis against variously prepared BSA with an anti-Glu antibody on nitrocellulose membranes (A-G). BSA-dotted membranes were treated with glutaraldehyde/Glu (A,B) or glutaraldehyde (C,D), or without treatment. BSA was assumed to be a binding dot pattern at the upper area of the line in each square. It was blocked with gelatin and incubated with (A,C,E) or without (B,D,F) the anti-Glu antibody. (G) Immunopositive control to show the binding of BSA as a dot pattern on the membrane with the anti-BSA antibody. Only after treatment with both glutaraldehyde and Glu (A), the immunoreactivity (IR) is obtained. (H,I) Glu-IR by attaching Glu to glass slides and immunostaining with (H) or without (I) the primary anti-Glu antibody (Primary) at higher magnification under a fluorescence microscope. Asterisks in (H) and (I) indicate the spot areas where the Glu was attached to glass slides. Bar = 50 μm.

Glu-IR in Retinas Prepared With Glutaraldehyde During FS

It was thought that the efficiency of paraformaldehyde linking of amino acids was not as high as that of glutaraldehyde. Therefore, to evaluate the effect of glutaraldehyde on the intensity of Glu-IR during the FS step, various concentrations of glutaraldehyde were added to the FS solution (Figure 4). The tissue sections were treated with BSA before the primary anti-Glu antibody incubation (Figures 4A–4H). In the samples prepared with 0.14% glutaraldehyde, the Glu immunolocalization was similar to that obtained with paraformaldehyde in the FS solution (Figure 4D). However, the Glu-IR became weaker with increasing glutaraldehyde concentrations (Figures 4F and 4H). This result also indicated that Glu is localized in the IS, probably reflecting the living state.

After treatment with paraformaldehyde or glutaraldehyde on the thin sections of the retinal tissue samples that were freeze-substituted with acetone alone, Glu-IR was not detected (data not shown), indicating that the chemical fixation during FS is necessary to retain the Glu molecules in the tissue samples. In addition, microwave treatment of the samples prepared by FS with paraformaldehyde or glutaraldehyde did not change the Glu-IR (data not shown).

Finally, to examine whether 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 (Figures 4I–4L). In sections of the specimen prepared with IVCT followed by FS without chemical fixatives (Figure 4J), 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 (Figure 4L). 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.