Rabbit anti foxa2 antibody

Tissues were homogenized in a Teflon-glass potter filled with buffer and protease inhibitor cocktail (Cwbiotech, China). Samples were centrifuged at 12000 × g (15 min, 4°C) and protein levels were measured by BCA kit (Tiangen, China). Extracted proteins were denatured, electrophoresed, and transferred to PVDF membranes (Millipore, 0.45 μm). The membranes were then blocked in 5% nonfat milk at room temperature before incubating with rabbit anti-GlyRα1 antibody (1:1000,Abcam,UK), rabbit anti-ClC-2 antibody (1:200, Santa Cruz, USA), rabbit anti-ClC-3 antibody (1:200, Santa Cruz, USA), rabbit anti-FoxA2 antibody (1:1000, Cell Signaling, USA) or rabbit anti-beta actin (1:1000, Proteintech Group, China) antibody overnight (4°C). After washes in TBST, membranes were incubated (1 h, room temperature) in goat-anti-rabbit-HRP secondary antibody (1:2500, KPL Inc, USA). Results were detected by Pierce ECL Plus Western Blotting Substrate (Pierce, USA) on Kodak X- ray film.

Pieces of uterine tissues were embedded in O.C.T. Compound and frozen immediately in liquid nitrogen. Air-dried tissue sections of 5 μm thickness were fixed for 5 min in acetone at –20 C. Nonspecific binding was blocked using 2% (v/v) goat serum in PBS (blocking buffer) for 30 min. Sections were incubated at 4 C overnight with rabbit anti-FOXA2 antibody (1:800; #3143, Cell Signaling Technology, Beverly, MA, USA) and mouse anti-Cytokeratin antibody (1:100; C2931, Sigma-Aldrich, St. Louis, MO, USA) diluted in blocking buffer. Nuclei were stained with Hoechst 33342 (Calbiochem, La Jolla, CA, USA). After washing with PBS, they were incubated for 1 h at room temperature with the secondary Goat Anti-Mouse IgG (H+L), F(ab’)2 Fragment (Alexa Fluor 488 Conjugate) antibody (1:500; Cell Signaling Technology) and Alexa Fluor 594 Goat Anti-Rabbit IgG (H+L) antibody (1:500; Invitrogen, Carlsbad, CA, USA) diluted in blocking buffer. Sections were subsequently washed in PBS and mounted
with Mount-Quick Aqueous (Daido Sangyo, Tokyo, Japan). Immunostaining was detected under a fluorescence microscope (Nikon, Tokyo, Japan).