Fig 1: Visual cycle proteins were abundantly expressed in the RPE flatmounts during the first 3 days in culture, but gradually decreased over time. RPE flatmounts obtained from 2- and 9-month-old wild-type C57BL/6J mice were cultured in complete medium for up to 7 days. RPE flatmounts treated with H2O2 were cultured for 16 h. (A) Protein lysates were prepared from the RPE flatmounts and subjected to western blotting analysis to detect the levels of visual cycle proteins RPE65 and CRALBP. Statistical analysis was performed using one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. n = 3. (B) RPE65 and CRALBP protein levels were also measured by immunofluorescence. Data from both the western blotting and immunofluorescence showed the gradual reduction in the two proteins in RPE flatmounts over time. Scale bar = 20 µm.