Erβ sc 8974

Ovaries and follicles were fixed in cold 4% paraformaldehyde, for 48 and 24 h, respectively, dehydrated in ethanol and toluene, embedded in paraffin and sectioned at 5 μm onto APES-treated microscope slides (ZLI-9001, Zhongshan Company, Beijing, China) for immunohistochemistry^{48 (link)} or immunofluorescence staining,^{49 (link)} as previously described. Cells were fixed in cold 4% paraformaldehyde for 20 min, membrane osmosed in 0.1% triton X-100 for 5 min, blocked with 10% normal donkey serum for 1 h. COV434 cells and human MGCs transfected with vectors were treated at adherent state and freshly isolated human MGCs were treated at suspension state. Primary antibodies used for immunohistochemistry were diluted as follows: ERα (sc-542, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and ERβ (sc-8974, 1:2000; Santa Cruz Biotechnology). Primary antibodies used for immunofluorescence were diluted as follows: ERα (sc-542, 1:100) and ERβ (ab3576, 1:200; Abcam, Cambridge, UK). An isotype-matched IgG was used as the negative control.

Dimethyl sulfoxide (DMSO), paraformaldehyde, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), Triton X-100, propidium iodide (PI), DNAse-free RNAse A, perfluorooctanoic acid, cholera toxin (CT), insulin, 3-(4,5-dimethyl-2-yl)2,5-diphenyl-2H-tetrazolium bromide (MTT), epidermal growth factor (EGF), hydrocortisone and sulforaphane were obtained from Sigma-Aldrich (St Louis, MO, USA). Horse serum, penicillin–streptomycin (P/S), Dulbecco’s Phosphate-Buffered Saline (PBS), Dulbecco’s Modified Eagle’s Medium (DMEM), trypsin solution (0.05%) were obtained from Gibco (Invitrogen, Paisley, UK). p53 monoclonal (DO-7), CDK6 monoclonal (75B9), CDK4 monoclonal (DCS-31) and p21 monoclonal (R.229.6) antibodies were obtained from ThermoFisher Scientific (Rockford, IL, USA). P27 Kip1 (D69C12) and cyclin D1 (92G2) antibodies were obtained from Cell Signaling (Danvers, MA, USA). The secondary antibodies Alexa-Fluor 555 goat anti-mouse IgG or 488 goat anti-rabbit IgG, and the blocking agent (normal goat serum) were obtained from Molecular Probes, Invitrogen. Matrigel Basement Membrane Matrix was obtained from Corning (New York, NY, USA). ER α (sc-8002) and ERβ (sc-8974) monoclonal antibodies were obtained from Santa Cruz Biotechnology (Bergheimer, HD, DE). ICI 182,780, GW 6471 was obtained from Tocris Bioscience (Avonmouth, Bristol, UK).

Dimethylsufoxide (DMSO), pharaformaldehyde, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), Triton X-100, Propidium iodide (PI), DNAse-free RNAse A, Perfluorooctanesulfonic acid potassium salt (PFOS), cholera toxin (CT), insulin, epidermal growth factor (EGF), and hydrocortisone were obtained from Sigma-Aldrich (St Louis, MO, USA). Horse serum, penicillin–streptomycin (P/S), Dulbecco’s Phosphate-Buffered Saline (PBS), Dulbecco’s Modified Eagle’s Medium (DMEM), and trypsin solution (0.05%) were obtained from Gibco (Invitrogen, Paisley, UK). p53 monoclonal (DO-7), CDK6 monoclonal (75B9), CDK4 monoclonal (DCS-31), and p21 monoclonal (R.229.6) antibodies were obtained from ThermoFisher Scientific (Rockford, IL, USA). P27 Kip1 (D69C12) and Cyclin D1 (92G2) antibodies were obtained from Cell Signaling (Danvers, MA, USA). The secondary antibodies alexa-fluor 555 goat anti-mouse IgG or 488 goat anti-rabbit IgG, and the blocking agent (normal goat serum) were obtained from Molecular Probes, Invitrogen. Matrigel Basement Membrane Matrix was obtained from Corning (New York, NY, USA). ERα (sc-8002) and ERβ (sc-8974) monoclonal antibodies were obtained from Santa Cruz Biotechnology (Bergheimer, HD, DE). ICI 182,780 was obtained from Tocris Bioscience (Avonmouth, Bristol, UK).