H 1500 10

Pan02 cells were incubated with 200 nM Cy5.5-conjugated atezolizumab, VEGF-Grab (negative control), or Ate-Grab for 6 h at 37 °C. After incubation, Pan02 cells were washed three times with PBS, fixed in 4% paraformaldehyde for 20 min at room temperature, and counterstained with DAPI (VECTOR, H-1500-10). Images were acquired using a custom-designed video-rate laser-scanning microscope.

Following enucleation, the cornea and lens were carefully removed under a dissecting microscope while maintaining the shape of the eyecup. The eyecup was fixed with 4% paraformaldehyde in PBS (Santa Cruz Biotechnology, no. 30525-89-4) for 2 h and washed with 5% sucrose in PBS three times for 5 min. The eyecup was dehydrated with 20% sucrose in PBS, embedded in 20% sucrose in O.C.T. (1:2 volume ratio, Sakura, no. 4583), and then flash-frozen for cryosectioning at 10 μm thickness. For immunostaining, cryosections were first blocked with 5% normal donkey serum in 0.2% Triton X-100 in PBS, and then incubated with primary antibodies overnight at 4 °C. The S-opsin and M-opsin antibodies were identical to those used for retinal flatmount staining. Cone arrestin was probed with polyclonal rabbit anti-cone arrestin antibody (1:400; Millipore Sigma, no. AB15282). Cone sheaths were stained with fluorescein-conjugated peanut agglutinin (1:200; Vector Laboratories, no. FL-1071). Secondary antibodies were identical to those used for retinal flatmount staining. After incubation for 2 h at room temperature with secondary antibodies, cryosections were washed three times for 5 min each before mounting with a mounting medium containing DAPI (Vector Laboratories, no. H-1500-10) and securing with a coverslip.

Following the blocking in 3% BSA and 0.3% Tx-100 in PBS for 60 min at RT, sections were washed in PBS for 5 min and incubated for 1 min in TrueBlack lipofuscin autofluorescence quencher (Biotium #23,007) in 70% ethanol. The sections were washed in PBS (3 × 5 min) and incubated in primary antibodies overnight at 4 °C. For the primary antibody details and their optimized IF settings, see Supplementary Tables 4 and 5. After rinsing in PBS, the sections were incubated in secondary antibodies for 1 h at RT in the dark and washed in PBS. Where applicable, the sections were then incubated in biotinylated primary antibody solution at 4 °C overnight, rinsed in PBS, incubated in streptavidin secondary antibody solution for 1 h at RT in the dark, and rinsed in PBS. The slides were mounted using an aqueous mounting medium with DAPI (Vector Laboratories #H-1500-10). Imaging was carried out on a Leica DM5500 B upright microscope (RRID:SCR_020219), and image analysis on Leica Application Suite X (RRID:SCR_013673).