Anti foxo3

The isolated ovaries were fixed and permeabilized with 4% PFA and then prepared for paraffin section. After dewaxing and rehydration of paraffin sections, membrane permeation was performed after washing. Then the sections were blocked with blocking buffer (5% goat serum, 0.3% Triton™ X-100 in 0.1 M PBS) for 1 h at room temperature, followed by the incubation with primary antibody overnight at 4 °C. After washing with TBST, sections were incubated with the secondary antibody at 37 °C for 1 h. After washing again, DAPI was added to probe the nuclei and mounted with a mounting medium (H-1400, Vector Laboratories, Inc). Axio Scan.Z1 (Zeiss) and Zeiss LSM780 were used for widefield and confocal imaging, respectively. Images were captured and analyzed using software Zeiss Zen (v2.1) and Image J (v1.52i). The primary antibodies used for immunostaining included: anti-DDX4 (1:200, ab13840, Abcam), anti-AMH (1:400, sc-6886, Santa Cruz Biotechnology), anti-cleaved CASP3 (9664, CST), anti-Foxo3 (1:250, Santa Cruz Biotechnology), and anti-BDNF (1:100, Abcam, ab108319). The secondary antibodies included: Alexa Fluor 546 conjugated goat anti-mouse (Thermo Fisher Scientific, A-11030, 2 μg/ml) and Alexa Fluor 488 conjugated goat anti-rabbit (Thermo Fisher Scientific, A-11008, 2 μg/ml).

Cells were seeded on coverslips, fixed with 3.7% formaldehyde and permeabilized with 0.5% Tween20, stained with primary anti-FOXO3 (Santa Cruz), anti-RRM2B (Rockland), γ-H2AX (Active Motif) and secondary antibodies, washed, stained with DAPI as nuclear control, and then mounted onto the slides as described [45 (link)]. The slides were analyzed by Immunofluorescence microscope system. Scale bars indicate 10μM.

The primary antibodies were anti-Lhx8 (1:500), anti-FOXO3 (1:100, Santa Cruz Biotechnology, USA), poly(ADP-ribose) polymerase (PARP)Antibody (Cell signaling, USA), and Antibody forLH-R (Acris antibodies Inc., USA). The secondary antibodies were Alexa 488 goat anti-rabbit and Alexa 546 goat anti-guinea pig (1:400, Invitrogen, UK). Actin and DAPI (Invitrogen, UK) were used to control the process of immune fluorescence. After staining, the sections were mounted. The staining was observed and images were obtained with a LSM 510 META confocal laser-scanning microscope (Carl Zeiss, Germany) or an epifluorescence microscope (Axio Imager 2, Carl Zeiss) with the ZEN image program.