Af0146

Oocytes were fixed in 4% paraformaldehyde for 30 min, and then permeabilized in permeate (DPBS containing 1% Triton X-100) for 20 min at room temperature. After blocking oocytes with 1% BSA for 1 h at room temperature, they were exposed to primary antibody at 4°C overnight, and washed three times for 10 min each in wash buffer (DPBS containing 0.01% Triton X-100 and 0.1% Tween 20), then stained with fluorescently labeled secondary antibodies and incubated at 37°C for 1 h. After incubation, oocytes were washed three times in washing buffer for 10 min each. Finally, oocytes were transferred to slide containing VECTASHIELD mounting medium with DAPI and sealed with glass cover and photographed under fluorescence microscope and intensity measured as described above.
Antibodies and dilution ratios used in immunofluorescence were as follows: Drp1 antibody (Affinity, DF7037, 1:300); Drp1(Ser616) antibody (Affinity, AF8470, 1:300); TOMM20 antibody (Affinity, DF4179, 1:200); Cytochrome C antibody (Affinity, AF0146, 1:100); CoraLite488- conjugated Goat Anti-Rabbit IgG (Proteintech, SA00013-2, 1:300).

The OC tumor tissue and cells were extracted to obtain the protein, and the protein was quantified by using the BCA kit. The 50 μg protein was loaded quantitatively and separated by SDS-PAGE with 10% separation gel. Electrophoresis conditions were as follows: 80 V (30 min) and then 120 V; electrophoretic transfer condition was as follows: 90 min, 200 mA, and transfer protein to the PVDF membrane; it was then sealed for 2 h in skim milk powder solution; the corresponding primary antibodies anti-NF-κB p65 (ab16502, Abcam, Cambridge, 1 : 500), anti-p38 (Abcam, ab31828, 1 : 1000), anti-mTOR (phospho S2448)(Abcam, ab109268, 1 : 1000), phospho-PI3K p85 (AF3242, Affinity, 1 : 500), anti-AKT (phospho T308) (ab38449, Abcam, 1 : 500), anti-Bcl-2 (Affinity, AF6139, 1 : 1000), anti-Bax (Affinity, AF0120, 1 : 1000), anti-Cyt-C (Affinity, AF0146, 1 : 1000), anti-caspase-3 (Affinity, AF6311, 1 : 1000), anti-cleaved caspase-3 (Affinity, AF7022, 1 : 1000), and anti-GAPDH (ab8245, Abcam, 1 : 500) were added proportionally and incubated overnight at 4°C. After washing the membrane, a second antibody was added and incubated at room temperature for another 1 h. After washing the membrane again, the developer and fixative 1 : 1 were mixed; then, the protein strip was developed with a gel imaging system, and the statistical results were analyzed.