Immunocytochemistry was performed as previously described [36 (link)–38 (link)]. Cells were fixed with 4% paraformaldehyde at room temperature (RT) for 10 min, and for intracellular markers, permeabilization of cells was carried out with 0.2% Triton X-100 at RT for 10 min. Next, cells were blocked in 10% donkey serum at RT for 30 min and then incubated in the primary antibodies at RT for 1 h or at 4 °C overnight. Primary antibodies were applied in this study: CD117 (R&D Systems, Cat. AF1356, 1:50), caudal type homeobox 2 (CDX2; Abcam, Cat. Ab76541, 1:200), aquaporin 5 (AQP5; EMD Millipore, Cat. AB15858, 1:100), prosurfactant protein C (SPC; Abcam, Cat. ab40879, 1:100), tight junction protein-1 (ZO-1; Thermo Fisher Scientific, Cat. 40-2200, 1:400), cardiac troponin T (cTnT; Abcam, Cat. ab125266, 1: 200), sarcomeric α-actinin (Sar α-actinin; Sigma Cat. A7811, 1:200), rhodopsin (Rho; Abcam, Cat. ab3267, 1: 200), recoverin (Rec; EMD Millipore, Cat. AB5585, 1: 500), arrestin 1 (Arr1; Abcam, Cat. Ab32099, 1:200). Second antibodies were conjugated with fluorescence, then were incubated with samples at 37 °C for 1 h. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; 1:1000 in PBS) at 37 °C for 10 min. Images were analyzed using fluorescence microscopy or a confocal microscopy system (Olympus).
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