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HESCs grown in 24-well cell culture plates, and endometrial tissue were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (w/v) for 20 min at room temperature. After three 5-min washes with PBS, the fixed coverslips were permeabilized in PBS with 0.1% Triton X-100 for 5 min at room temperature. Nonspecific sites were blocked in PBS for 1 h at 37 °C. The cells were incubated with fluorescein isothiocyanate-labeled phalloidin (1:200; P5282, Sigma-Aldrich) and an anti- flag antibody (1:200; 8146; Cell Signaling Technology; RRID: AB_10950495) at 4 °C overnight, the endocytic function was determined using anti-Rab11 (1:200; 5589S; Cell Signaling Technology; RRID: AB_10693925), anti-LRP5(1:100; sc-390267; Santa Cruz),and anti-LRP6 (1:100; sc-25317; Santa Cruz; RRID: AB_627894). After washing with BSA/PBS (three times), the coverslips were further incubated with Alexa Fluor 594-conjugated donkey anti-goat IgG (1:200, Invitrogen; RRID: AB_141359) and Alexa Fluor 488-conjugated goat antirabbit IgG (1:200, Invitrogen; RRID: AB_141607). Nuclei were stained with 4′, 6-Diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich). Finally, images were captured by fluorescence confocal microscopy (Olympus, FV10i).