hESCs were grown on Matrigel-coated coverslips. Cells were fixed with the 4% paraformaldehyde solution containing 0.5% Triton X-100. Fixed cells were incubated with the blocking solution (3% BSA in PBS containing 0.1% Tween 20) for 1 h. Cells were then treated with primary antibodies diluted in the blocking solution and incubated overnight at 4C. Primary antibodies used for staining included anti-ZO-1 (Invitrogen), anti-SOX2 (Abcam), anti-NANOG (Thermo Fisher Scientific), anti-OCT3/4 (Santa Cruz Biotechnology), anti-SOX1 (Cell Signaling), anti-KI67 (Abcam), anti-γ-tubulin (Abcam), anti-acetylated tubulin (Sigma-Aldrich), anti-ARL13B (Proteintech).
The slides were then washed with PBS containing 0.1% Tween 20, stained with secondary antibodies and DAPI and mounted with the vectashield antifade mounting medium (Vector Laboratories). The slides were viewed with a DeltaVision microscope (GE healthcare). Sum stack of image slices was used for intensity quantification. ImageJ was used to determine the signal intensities of nuclei. Normalized signal intensities were calculated by subtracting background signals from nuclear signals.
The slides were then washed with PBS containing 0.1% Tween 20, stained with secondary antibodies and DAPI and mounted with the vectashield antifade mounting medium (Vector Laboratories). The slides were viewed with a DeltaVision microscope (GE healthcare). Sum stack of image slices was used for intensity quantification. ImageJ was used to determine the signal intensities of nuclei. Normalized signal intensities were calculated by subtracting background signals from nuclear signals.