Sc 376279

Whole cell lysates were prepared in NETN buffer containing 20 mM Tris HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA and 0.5% Nonidet P-40, separated on SDS-PAGE gels, and transferred to PVDF membranes. Western blotting was performed using the appropriate primary antibodies against NEK2 (1:200, sc55601, Santa Cruz Biotechnology), Wnt1 (1:500, ab15251, Abcam), Wnt4 (1:500, sc376279, Santa Cruz Biotechnology), β-catenin (1:1000, #8480, Cell Signaling Technology), Flag (1:1000, F1804, Sigma) and GAPDH (1:1000, #5174, Cell Signaling Technology) overnight at 4 °C. The PVDF membranes were then incubated with secondary antibodies and detected via enhanced chemiluminescence.

Paraffin-embedded section of decidual tissues were dewaxed using dimethylbenzene and rehydrated in ethanol at different concentrations (100%, 95%, 90%, 80%, 70% and 50%). For immunofluorescence, the sections were blocked with 10% donkey serum after antigen retrieval using citrate sodium solution, and then they were incubated with primary antibodies (anti- Rev-erbα (sc-393215, Santa Cruze); anti-Vimentin (ab92547, Abcam), anti-Wnt4 (sc-376279, Santa Cruze)) overnight at 4 ℃. The sections were incubated with secondary antibodies for 2 h at room temperature after washed three times with tris-buffered saline (TBS) (10 min each), followed by 4′,6-diamidino-2-phenylindole (DAPI) staining. Mean gray value was calculated using ImageJ software. Relative mean gray value = mean gray value of cells /the mean value of mean gray value of cells from human/mouse with normal sleep. For hematoxylin–eosin (HE) staining, the sections were stained with hematoxylin solution for 5 min, and then washed with ultrafiltration water for 5 s. Next, the sections were stained with eosin solution for 3 min and dehydrated in ethanol at different concentrations (50%, 70%, 80%, 90%, 95% and 100%) and dimethylbenzene in turn. The slides were sealed with mounting medium and taken pictures using a fluorescence microscope.