Photoreceptor OS were obtained from InVision BioResources (WA, USA) or prepared in-house according to Molday et al., 198739 (link). OS labeling: Two mg/ml stock solution of FITC isomer 1 (F7250, Sigma) in 0,1M sodium bicarbonate at pH 9.0–9.5, was prepared, filter-sterilized, and stored in aliquots at −20 °C. For labeling with FITC, OS were suspended in solution containing 10% sucrose, 20 mM sodium phosphate, and 5 mM taurine, and incubated with FITC for 1,5 h at room temperature, rotating in the dark. FITC-labeled OS were washed 5 times in buffer containing 10% sucrose, 20 mM sodium phosphate and 5 mM taurine, suspended in DMEM, aliquoted and stored at −80 °C until use.
iPSC-RPE cells were incubated with OS for 2 h/37 °C, after which the OS were removed by triple washing with PBS. Cells were fixed by 4% PFA for 15 min/RT, washed and permeabilized by 0.1% Triton-X. Phallodin staining was performed for 30 min, the samples were then mounted with vectashield mounting media with DAPI. The samples were visualized by Leica confocal microscope TCS SP5 using HCX PL APO lambda blue 63X/ 1.4 OIL objective.
iPSC-RPE cells were incubated with OS for 2 h/37 °C, after which the OS were removed by triple washing with PBS. Cells were fixed by 4% PFA for 15 min/RT, washed and permeabilized by 0.1% Triton-X. Phallodin staining was performed for 30 min, the samples were then mounted with vectashield mounting media with DAPI. The samples were visualized by Leica confocal microscope TCS SP5 using HCX PL APO lambda blue 63X/ 1.4 OIL objective.