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

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 (-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.

Extracellular space in mouse cerebellar cortex revealed by in vivo cryotechnique

“…The method has been applied to various organs of living mice to reveal their functional morphology under physiological or pathological conditions (Ohno et al, 2004a;Terada et al, 2006a). Although the in vivo cryotechnique has several technical advantages, such as antigen retrieval effects , detection of rapidly changing signal molecules (Terada et al, 2006b), and effective preservation of soluble molecules Li et al, 2006) for immunohistochemistry at a light microscopic level, one important benefit is the in vivo freezing of target organs in living animals without tissue resection or stopping blood circulation (Ohno et al, 2004a). Therefore, functionally significant structures of living cells and tissues changing under various hemodynamic conditions can be directly captured by IVCr, and the artifacts due to anoxia after tissue resection can be minimized .…”

Raman Microscopy of Freeze‐dried Mouse Eyeball‐slice in Conjunction with the “in vivo Cryotechnique”

“…The eyeballs were immediately frozen with the liquid isopentane-propane cryogen (À1938C) (Fig. 1a, step 1) (Terada et al, 2006b). The mice were maintained under normal physiological conditions with their hearts beating (Ohno et al, 1996).…”

“…Freshly prepared mouse rhodopsin protein was purified as reported previously (Ohguro et al, 1995;Terada et al, 2006b). Human hemoglobin protein was purchased form Sigma (#H7379; St. Louis, MO).…”

“…Human hemoglobin protein was purchased form Sigma (#H7379; St. Louis, MO). The proteins were dissolved in phosphate buffered saline (pH 7.4) and put on glass slides under the room light at room temperature, and quickly frozen by rapid emersion in an isopentane-propane mixture cooled in liquid nitrogen (Terada et al, 2006b). The frozen samples were freeze-dried and their Raman spectrum was recorded and analyzed, as described above.…”

“…Since the Raman spectra was acquired after quick-freezing and freeze-drying of the sample and the subsequent fixing and staining of the embedded section do not affect the Raman spectra. A freezesubstitution step is also possible after freeze-drying and the Raman spectra are acquired if an immunohistochemical study is desired (Terada et al, 2006b).…”