Decidualization of hESCs Visualized

hESCs grown in 8-well chambers (Millipore, Billerica, MA, USA) were exposed to a decidualization stimulus of 8-Br-cAMP plus MPA for the indicated durations and then fixed with 4% paraformaldehyde (w/v) for 30 min at room temperature. Next, the cells were washed with PBS and permeabilized with 0.5% Triton X-100 in PBS at room temperature. Subsequently, the cells were blocked with 3% BSA in PBS and incubated with fluorescein isothiocyanate-labeled phalloidin (1:300; P5282, Sigma, St. Louis, MO, USA) at 4 °C overnight. Cell nuclei were stained with DAPI (5 μg/ml) on the following day. Finally, the cells were visualized using a confocal microscope (Leica, Wetzlar, Germany) [24 (link)].