Rabbit anti rpe65

Mice were euthanized 12 weeks after injection. For frozen cross sections, eyes were removed, fixed in 4% paraformaldehyde (PFA)/PBS at RT for 1 hr, and then washed three times in PBS for 5 min each. The center of the cornea was cut off before cryoprotection in 30% sucrose/PBS overnight at 4 °C. Then the eye cups were embedded in O.C.T. (Sakura Finetek USA, Inc.). After rinsing with PBS for 5 min, frozen retinal slices with a thickness of 10 μm were blocked with 5% normal goat serum and 0.2% Triton X-100 in PBS for 1 hr at RT. The slices were then incubated with primary antibodies in blocking solution overnight at 4 °C. Rabbit anti-RPE65 (1:250 dilution; Abcam), rabbit anti-S opsin (1:50 dilution; Novus Biologicals), and rabbit anti-M opsin (1:1000 dilution; Novus Biologicals) were the primary antibodies used in this study. After washing 3 times in PBST for 10 min each, slices were treated with goat anti-rabbit IgG Alexa Fluor 594 (1:1000 dilution; Abcam) for 1 hr at RT. After 3 washes in PBS for 10 min each, slices were stained with DAPI and photographed using a confocal laser microscope (Nikon, Tokyo, Japan).