Fig. 35.1
(a) Flow chart of procedure with IVCT and FS for immunohistochemical detection of phosphorylated protein. Dynamically changing molecule under phosphorylation is immediately immobilized with IVCT. (b) Schematic representation of IVCT for eyeballs of living mouse. (c, d) HE staining (c) and immunostaining for phosphorylated rhodopsin (d) in mouse retina with IVCT. ON outer nuclear layer, RC rod and cone photoreceptor cell layer. Rhodopsin is immunostained in outer segment (arrows in c, d) but not in inner segment (asterisks in c, d). (e) Schematic representation of protocol (left lane) and light micrographs of DIC (middle lane) and phosphorylated rhodopsin immunostaining (right lane) images of retinas exposed to strong light for 10, 30, 60 and 180 s after the dark adaptation. Arrows indicate rod and cone layer s (RC). PE pigment epithelium, Ch choroids. Precise data have been reported in the previous paper (Terada et al. [16]). Bars 20 μm
35.3 IVCT of Mouse Eyeballs Under Various Illumination Conditions
IVCT was performed against left eyeballs of mice under deep chloroform anesthesia, which were kept under different illumination conditions (Fig. 35.1b). To treat the eyeballs more easily than those in the previous cornea experiment with IVCT [17–19], palpebral skins were slightly cut around them. The eyeballs were then immediately frozen in vivo by pouring liquid isopentane –propane cryogen (−193 °C), which was cooled in liquid nitrogen just before pouring [1, 17–19]. For complete darkness, after the 12-h dark adaptation, the IVCT was performed under infrared light by directly observing the mice with an infrared camera (Fig. 35.1b). For the condition under the safety red light, IVCT was performed under the light shielded by a red-color plate for 2 min after the dark adaptation. To obtain light-illuminated mouse retinas, the dark-adapted eyeballs were continuously illuminated with a flashlight for 10, 30, 60, and 180 s before performing IVCT. They were then immediately frozen in a similar way with the liquid cryogen and maintained in liquid nitrogen at a low temperature . Frozen eyeballs were finally removed with a pair of nippers in the liquid nitrogen and then processed for the next freeze-substitution step.
35.4 Immunoreactivity of P-Rho in IVCT Retina Under Various Illumination Conditions
In the eyeballs prepared with IVCT the eyeballs were always frozen from the scleral outside with the poured cryogen, and so the rod and cone layer was well preserved at a light microscopic level (Fig. 35.1c). Under daylight condition, the rod and cone layer was specifically immunostained with the anti-P-Rho334 antibody (Fig. 35.1d).
P-Rho334 immunoreactivity was completely eliminated after the dark adaptation. In the living animal retina , phototransduction has been thought to proceed in time courses of milliseconds to seconds [7]. Whereas the P-Rho334 immunoreactivity was not detected in the living mouse retina after the 10-s light exposure (Fig. 35.1e), it was definitely recovered in retinas exposed to visible light for 30 s and more (60 and 180 s) (Fig. 35.1e).
35.5 Concluding Remarks
IVCT revealed that 334Ser rhodopsin in retinal tissues of living mice is generally phosphorylated between 10 and 30 s after visible light stimulation.
