Af2644

Granulosa, COCs, ovarian stromal tissue, or whole ovaries were lysed in RIPA buffer (Boston BioProducts). Proteins were separated using a 10% Bis-Tris gel and transferred to an immobilon-P membrane (Millipore). Primary antibodies used included GAS1 (1:1000 dilution, AF2644; R&D system) and AKT (1:2000 dilution, 9272; Cell Signaling Technology). Signals were detected using the Peirce ECL 2 Western Blotting Substrate (Thermo Scientific).

Ovarian tissues were fixed in 4% paraformaldehyde and either embedded in paraffin for routine histological (hematoxylin and eosin [H&E]) and immunofluorescent (IF) analyses or frozen in optimal cutting temperature compound (Ted Pella, Inc.) for other specific IF analyses. Primary antibodies specific for GAS1 (AF2644; R&D system), PTX3 (P0496; Sigma Aldrich), SFRP4 (HPA009712; Sigma Aldrich), PECAM-1 (553373; BD Pharmingen), and collagen IV (ab6586; Abcam) were used in the IF studies. Cell nuclei were counterstained with 5μg/ml Hoechst 33342 dye (Sigma Aldrich). Digital images of H&E and IF staining were captured using a Zeiss AxioPlan2 microscope in the Integrated Microscopy Core, and images of IF staining were captured with Zeiss LSM780 confocal microscope in the Optical Imaging and Vital Microscopy Core at Baylor College of Medicine.