Cells were fixed in 10% formamide for 15 min at room temperature after each day of differentiation. Then, their membranes were permeabilized by PBS containing 0.25% Triton X-100 (Sigma-Aldrich, MA, USA) for 10 min. Cells were blocked with Tris-buffered saline-tween 20 (TBST) containing 1% bovine serum albumin (BSA; Sigma-Aldrich, MA, USA) for 30 min, primary antibodies (Millipore, MA, USA) for 1 h and Alexa 488-conjugated secondary antibody (Cell Signaling Technology, MA, USA) for 1 h in the dark. Then, 4,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, MA, USA) was applied, and fluorescence images were obtained via confocal microscopy (Nikon, Japan)68 (link).
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