Anti rpe65

At the indicated time points after the subretinal injection, mice were sacrificed and RPE-choroid-scleral complexes were prepared. The complexes were then treated with anti-FLAG (catalog no. MA1-142-A488, Thermo Fisher Scientific), anti–ZO-1 (catalog no. 339194, Thermo Fisher Scientific), or anti-RPE65 (catalog no. NB100-355AF488, Novus Biologicals) antibodies and observed under a confocal microscope. The nuclei were identified using 4′,6-diamidino-2-phenylindole (catalog no. D9542, Sigma-Aldrich).

Cross-sections of the retina or brain and whole-mount retinas for immunohistochemical analysis were firstly washed several times with PBS, and then blocked in PBS containing 5% goat serum and 1% Triton x-100 for 1h at room temperature. The blocked tissues were incubated with the following primary antibodies overnight at 4°C: anti-β-amyloid, clone 6E10 (1:500; BioLegend), anti-β-amyloid, clone 4G8 (1:500; BioLegend), anti-rhodopsin, clone 1D4 (1:1000; Santa Cruz), anti-M-opsin (1:500; Millipore), anti-S-opsin (1:500; Millipore), anti-PKCα (1:200; Abcam), anti-PKCα (1:100; Invitrogen), anti-CtBP2 (1:200; BD), anti-PSD95 (1:100; CST), anti-ZO-1 (1:100; Invitrogen), anti-RPE65 (1:200; Novus), anti-p16^ink4α (1:100; Proteintech), anti-p21 (1:100; Santa Cruz), anti-cleaved caspase3 (1:100; CST), anti-RIPK3 (1:100; Santa Cruz), anti-pRIPK3 (1:400; CST) and anti-pMLKL (1:100; Abcam). AlexFluro-488 or -555 conjugated secondary antibody (1:500; CST) incubation were performed for 2.5 h at room temperature. Thereafter, DAPI (1:1000, Invitrogen) were used to label the nuclear for 10 min at room temperature.