Ab202063

Protein extraction was performed by washing the cell pellet with ice-cold PBS. Next, 1 mL of ice-cold RIPA lysis buffer (Sigma) and a protease inhibitor cocktail (Thermo Scientific, Rockford, IL, USA) were added and incubated on ice for 30 min. Then, 30 µg of cell lysates were resolved on 4–20% ExpressPluS PAGE Gel (GenScript, Piscataway, NJ, USA) following concentration measurements. The eBlot Protein Transfer device (GenScript, Piscataway, NJ, USA) was used to transfer the proteins onto a PDVDF Transfer Membrane (Thermo Scientific, Rockford, IL, USA). The membranes were washed and incubated for 1 h with a secondary anti-mouse antibody conjugated with HRP (Cell Signaling Technology, Danvers, MA, USA). Pierce ECL Western blotting Substrate (Thermo Scientific, Rockford, IL, USA) and BioRad Universal Hood II with a Chemiluminescence System (BioRad, Hercules, CA, USA) were used to visualize the result. Antibodies: Recombinant Anti-FANCD2 antibody [EPR2302] (abcam—ab108928) 1/1000; Recombinant Anti-Rad51D antibody [EPR16205] (abcam—ab202063) 1/1000; Recombinant Anti-β Actin antibody [EPR21241] (abcam—ab213262) 1 µg/mL; Recombinant Anti-Rad51D antibody [EPR16205] (ab202063) 1/1000.

Immunofluorescence (IF) was carried out on ovarian tissue sections as follows. Briefly, after fixation with 4% paraformaldehyde for 12 h, according to the standard histological procedures the ovarian pieces were processed for paraffin sectioning. Serial 5µm sections were heated at 60 °C for 2 h. After this the slides were rehydrated with a series of graded ethanol and washed with PBS 0.01 M sodium citrate were used for antigen retrieval at 95°C. After blocking for 1 hr with 10% BSA, the slides were incubated overnight at 4 °C with primary antibodies, anti: VASA (Abcam, ab13840, USA), SCP3 (NOVUS, NB300-232), DAZL (Abcam, ab34139), ɤH2AX (Sigma, SAB4501369), RAD51 (Abcam, ab202063), MLH1 (Abcam, ab92312), diluted to optimal concentrations. After thoroughly rinsing with TBS, secondary antibodies (CY3, A0516, Beyotime; FITC, A0568, Beyotime, China) diluted 1:200 were applied at 37 °C for 1h in the dark. After washing three times with PBS, the samples were incubated with Hoechst33342 (Solarbio, China) and observed under a fluorescence microscope. Positive cells were scored as previously described [67 (link)]. Negative controls were performed omitting the primary antibodies after the blocking procedure (not shown).