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

Nobuo Terada ^1*, Nobuhiko Ohno ¹, Hiroshi Ohguro ¹, Zilong Li ¹ and Shinichi Ohno ¹

¹ Department of Anatomy, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Tamaho, Japan (NT,NO,ZL,SO), and Department of Ophthalmology, Hirosaki University School of Medicine, Hirosaki, Japan (HO)

^* To whom correspondence should be addressed. E-mail: nobuot{at}yamanashi.ac.jp.

Submitted on September 24, 2005
Accepted on 22 December 2005

	Abstract

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 (-196°C). The frozen whole-mount eyeballs were freeze-substituted in acetone containing paraformaldehyde and commonly embedded in paraffin wax. Deparaffinized sections were immunostained with anti-phosphorylated ³³⁴Ser Rho (P-Rho334) antibody. The immunoreactivity of P-Rho334 was specifically recognized in the outer segments of mouse retinas exposed to day light. In the 12 hours’ dark-adapted retinas, the 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, the P-Rho334 immunoreactivity was definitely recovered, as similar to that under the day light 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.

Key Words: in vivo cryotechnique, freeze-substitution, rhodopsin, phosphorylation, immunohistochemistry

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