Sc 10439

Total protein was extracted from follicles and roughly equal numbers of CLs with radioimmunoprecipitation assay buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium
deoxycholate, 0.1% sodium dodecyl sulfate) containing protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel
electrophoresis and electrophoretically transferred onto a polyvinylidene fluoride membrane (Hybond, Amersham Pharmacia Biotech, Piscataway, NJ, USA). Following transfer, the membranes were
incubated for 1.5 h in 5% nonfat dry milk at room temperature, then overnight at 4˚C with anti-SNAI3 polyclonal (1:500, sc-10439; Santa Cruz Biotechnology) and β-actin (1:5000, SC-47778;
Santa Cruz Biotechnology) antibodies. After washing thrice in Tris-buffered saline Tween 20, the membranes were incubated for 1 h at room temperature with the appropriate secondary antibody
in the same buffer. Finally, the proteins were detected using Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, Billerica, USA) with an enhanced chemiluminescence western
blotting analysis system (Tanon 5500 Multi, Shanghai, China). β-Actin levels were measured as the internal loading control. The relative intensities of the bands were analyzed by Quantiscan
software (Biosoft, Great Shelford, Cambridge, UK) and normalized to β-actin from the same blot.

Immunohistochemistry was carried out as described previously [25 (link), 26 (link)]. Briefly, the collected tissues were
immediately snap frozen in liquid nitrogen and stored at –80˚C. Ovaries were cryosectioned at 7 mm, fixed in acetone at –20˚C for 10 min, and incubated in 0.3% (v/v) Triton X-100 in
phosphate-buffered saline (PBS), pH 7.2, for 20 min. Endogenous peroxidase activity was quenched by incubating the samples in 3% (v/v) H₂O₂ for 20 min, followed by
washing with PBS. Sections were blocked with immunoglobulin G from the same animal species as the primary antibody for 1 h at room temperature (22–26ºC), and then incubated at 4˚C overnight
with the primary antibody to SNAI3 (1:20 dilution, sc-10439; Santa Cruz Biotechnology, Santa Cruz, CA USA). Thereafter, the samples were incubated with a horseradish peroxidase-conjugated
secondary antibody for 60 min at room temperature before being developed with a diaminobenzidine peroxidase substrate kit (ZLI-9033; ZSGB-BIO, Beijing, China). Negative controls were
incubated with pre-immune serum instead of the primary antibody. Finally, the sections were counterstained with hematoxylin, dehydrated, mounted, and digitally photographed using a
microscope (Olympus, Tokyo, Japan).

For immunofluorescence staining, embryos were fixed in 4% paraformaldehyde for 30 min and permeabilized in incubation medium (0.5% Triton X-100 in 3 mM MgCl₂, 20 mM HEPES, 50 mM
NaCl, 300 mM sucrose, and 0.02% NaN₃, pH 7.4) for 30 min (blastocysts for 40 min). This was followed by incubating with 1% bovine serum albumin in PBS for 30 min at room
temperature. The embryos were incubated with a goat anti-SNAI3 polyclonal antibody (1:50, sc-10439; Santa Cruz Biotechnology) overnight at 4˚C. After washing thrice in PBS for 5 min each,
the embryos were incubated with fluorescein-isothiocyanate-conjugated rabbit anti-goat IgG (1:100, sc-2777; Santa Cruz Biotechnology) for 60 min at room temperature. The embryos were then
washed thrice in PBS for 5 min each, stained with 10 μg/ml 4′,6-diamidino-2-phenylindole (Sigma-Aldrich), washed, and finally mounted on 1,4-diazabicyclo (2.2.2) octane
hydrochloride-containing (Sigma-Aldrich) glass slides. The mounted embryos were observed under a confocal laser scanning microscope (TCS SPE, Leica, Wetzlar, Germany).