Ib80826

RPTEC were seeded in a special 8 μ well-slide (IB80826) from Ibidi (Munich, Germany). The cells were fixed in 4% paraformaldehyde (KEBO, Oslo, Norway) in 1xPBS and incubated for 10 min on ice. The cells were permeabilized using 0.1% Triton X-100 (VWR International Oslo, Norway) in PBS for 5 min, followed by blocking in 3% BSA for 30 min and incubation with the appropriate primary and secondary antibodies for 60 and 30 min, respectively. Protein localization was detected using Zeiss-LSM510 Meta confocal microscopy (Oberkochen, Germany).

To generate embryoid bodies (EB), iPSCs were cultured with differentiation medium (50% F-12, 50% DMEM, 20% FCS, 1% ITS solution) in an ultra-low attachment six-well plate (ultra-low attachment surface plate, 3471; Corning, New York, NY, USA) for 8 days. The EBs were then transferred to gelatin-coated chamber slides (µ-slide eight-well, ibi-treat, ib80826; Ibidi, Martinsried, Germany) and cultured for a further 2 days to enable cellular attachment and differentiation. To investigate their differentiated states, the cells were washed with PBS and fixed with 4% phosphate-buffered paraformaldehyde for 30 min, and then washed three times with PBS. Thereafter, the fixed cells were incubated with PBS containing 3% BSA and 0.1% TritonX-100 for 30 min. For blocking and permeabilization, the plates were washed three times with 0.1% Triton X-100/PBS, and then incubated with each primary antibody at 4 °C overnight (Supplementary Table 3). After further washing three times with 0.1% TritonX-100/PBS, the cells were reacted with secondary antibodies at room temperature for 60 min.