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The purpose of this study is to analyze the time-dependent molecular states of rhodopsin (Rho) phosphorylation in the specimens originating from eyeballs cryoimmobilized in situ in living animals. Whole eyeballs of living mice under various dark- and light-exposure conditions were quickly frozen using the in vivo cryotechnique with isopentane-propane cryogen cooled down in liquid nitrogen (-196C). The frozen whole-mount eyeballs were freeze substituted in acetone containing paraformaldehyde and embedded in paraffin wax. Deparaffinized sections were immunostained with anti-phosphorylated (334)Ser Rho (P-Rho334) antibody. Immunoreactivity of P-Rho334 was specifically recognized in the outer segments of mouse retinas exposed to daylight. In the 12-h dark-adapted retinas, P-Rho334 immunoreactivity was completely eliminated. Moreover, in other retinas dark adapted for 12 or 36 hr and then exposed under the safety red light for 2 min, it was still barely recognized. Even in the eyeballs exposed to strong visible light for 10 sec, it was not detected. However, after 30, 60, and 180 sec of visible light exposure, P-Rho334 immunoreactivity was definitely recovered, similar to that under daylight condition. This is a new immunohistochemical approach to visualize the time-dependent Rho phosphorylation of living mice using the in vivo cryotechnique, in which changes could be detected within seconds following exposure to light.

Immunohistochemical Detection of Phosphorylated Rhodopsin in Light-exposed Retina of Living Mouse with In Vivo Cryotechnique