Dp72 camera

iRPE, fRPE and iCell RPE cells were cultured on 12 mm diameter transwells, (CLS3460, Sigma-Aldrich) coated with MRF (at a 1:30 dilution) in RDMsA for 42 days (unless stated otherwise) before processing for immunohistochemistry (see SM for detailed protocol). iPS on coverslips were processed for immunohistochemistry at around 60% confluence. Briefly, cells were fixed in 4% paraformaldehyde for 10-20 min at room temperature (RT), rinsed with PBS and blocked for 3-5 h in 5% foetal bovine serum + 5% normal goat serum + 0.1% Triton X-100 in PBS 1×. Primary antibody was incubated overnight at 4 °C in blocking solution (see SM for antibody references and dilutions). Primary antibody was washed with PBS 1X before secondary antibody incubation (see SM) 1 h at RT in PBS 1×. After wash with PBS 1×, the transwell membrane was cut and mounted in Mowiol. Z-stacks were acquired using a LSM700 confocal microscope from Zeiss at the EPFL BioImaging & Optics Core Facility (EPFL-BIOP). Other images were acquired with a BX60 microscope from Olympus equipped with a DP72 camera or a Leica DM6B microscope equipped with a DFC9000GT camera.

iRPE cells were cultured on coverslips or 12 mm diameter transwells (Sigma-Aldrich, Schaffhausen, Switzerland) and coated with MRF (at a 1:30 dilution) in RDMsA for 42 days before processing for immunostaining. Briefly, cells were fixed in 4% paraformaldehyde for 10–20 min at RT, rinsed with PBS and blocked for 3–5 h in 5% fetal bovine serum + 5% normal goat serum + 0.1% Triton X-100 in PBS 1X. Primary antibodies (Supplementary Table S21) were incubated overnight at 4 °C in blocking solution. Primary antibodies were washed with PBS before secondary antibody (Supplementary Table S21) incubation for 1 h at RT in PBS. After washing steps with PBS, transwell membranes were cut and mounted in Mowiol. Z-stacks were acquired using a LSM700 confocal microscope (Zeiss, Feldbach, Switzerland). For immunocytochemistry on cell sections, after cell fixation, transwell membranes were embedded on OCT compound and 7 µm sections were performed. Immunostainings were performed as described above. Images were acquired with a BX60 microscope from Olympus equipped with a DP72 camera or a Leica DM6B microscope equipped with a DFC9000GT camera and with confocal microscope.