Anti mouse igg hrp linked antibody

Primary antibodies targeting the following proteins were used: TSPAN9 (1:1000, Abcam), LC3 (1:1000, CST), P62 (1:1000, CST), Beclin1 (1:1000, Bioworld), PI3K (1:1000, CST), p-PI3K (1:1000, CST), AKT (1:1000, CST), p-AKT (1:1000, CST), mTOR (1:1000, CST), and p-mTOR (1:1000, CST). Cells were collected and lysed in ice-cold RIPA lysis buffer (1× Tris-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS). Then, 40 µg of cell lysate from each sample was separated by SDS-polyacrylamide gel electrophoresis (PAGE) through a 10% gel. Electrophoretic transfer of the proteins from the gels to nitrocellulose membranes was carried out in a transblotting chamber. After the membranes were blocked with 5% nonfat milk (w/v) in phosphate-buffered saline (PBS) for 1 h to inhibit nonspecific binding, they were incubated with primary antibodies at room temperature for 2 h. The membranes were then washed 3 times with PBST before secondary antibodies diluted in PBST were incubated with the membranes for 40 min at room temperature. The following secondary antibodies were used: anti-rabbit IgG HRP-linked antibody and anti-mouse IgG HRP-linked antibody (Sigma, 1:6000). The membranes were washed 6 times with PBST for 1 h, and the protein bands were visualized by chemiluminescence (Clarity Western ECL; Bio-Rad).

Total protein isolated from rat ovaries was used for Western blotting. Extracts containing equivalent quantities of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 7.5-12% gels) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The antibodies used included β-actin (Sigma-Aldrich, USA); PI3K, Akt, mTOR, PCNA, cleaved-caspase3, Bax, Bcl2, and phospho-CaMKII (Abcam, Britain); phospho-Akt, GDF9, BMP15, and BMPR2 (Abclonal, China); Smad3 (Beyotime, China); caspase3 (Bioss, China) and CaMKII (Cell Signaling Technology, USA). The protein band corresponding to protein specifically bound by the primary antibody was detected by an anti-rabbit or an anti-mouse IgG-HRP-linked antibody (Sigma-Aldrich, USA). Antibody-reactive bands were visualized using chemiluminescence (Sigma-Aldrich, USA).

Ovarian tissues were fixed in 4% paraformaldehyde (PFA), embedded in paraffin, and sliced into 5-µm sections. Every fifth slide was stained with hematoxylin and eosin (H&E) for differential follicle counts. The corpus luteum, cysts, antral follicles and atretic follicles were defined according to a previous study 62 .
Sample sections were processed for immunohistochemistry to assess GC proliferation. The sections were incubated with PCNA antibody (Abcam, Britain) overnight at 4 °C. The next day, the sections were incubated with an anti-mouse IgG-HRP-linked antibody (Sigma-Aldrich, USA) for 60 minutes at room temperature. Subsequently, the sections were visualized with a diaminobenzidine tetrahydrochloride (DAB) substrate chromogen system (Boster, China) at room temperature for 3 minutes. Negative controls were incubated with phosphate-buffered saline (PBS) instead of the PCNA antibody and showed no staining. The proliferation index of PCNA-positive cells was determined by the average number of positively stained GCs divided by the average total number of GCs in the area multiplied by 100.