Bovine serum albumin bsa

The prepared sections were blocked with 3% (w/v) bovine serum albumin (BSA; ZSbio) in PBST (0.1% [v/v] Triton X-100 in PBS) for 1 h at room temperature and then incubated with the following primary antibodies overnight at 4°C: anti-Sycp3 (1:200, Abcam, Cambridge, MA, USA), anti-synaptonemal complex protein 1 (Sycp1; 1:200, Abcam), anti-Vasa (1:500, Abcam), anti-c-kit (1:500, Abcam), anti-Shp2 (1:200, Santa Cruz Biotechnology), anti-Plzf (1:500, Santa Cruz Biotechnology), anti-cleaved caspase 3 (1:200, Cell Signaling Technology, Boston, MA, USA), anti-Dmc1 (1:200, Abcam), anti-Smc3 (1:500, Abcam), and anti-DNA repair recombinase rad51 (Rad51; 1:200, Invitrogen). After being washed three times with PBST, the samples were incubated with the following secondary antibodies at a 1:200 dilution for 1 h at 37°C: Alexa Fluor 594/488-labeled anti-rabbit or anti-mouse IgG (YEASEN, Shanghai, China). The slides were subsequently washed three times in PBST and mounted with Vectashield containing 4’-6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA).

Incomplete freund's adjuvant (IFA), PEG, horseradish peroxidase (HRP; H1759), sodium borohydride (NaBH₄; 10H3440), sodium m-periodate (NaIO₄) (38F-0860) and (+)-biotin-N-hydroxysuccinimide (NHSB) (HMBD0595 V) were from Sigma-Aldrich Co. (St. Louis, MO, USA). Hypoxanthine-aminopterin-thymidine (HAT) and hypoxanthine-thymidine (HT) were from Corning Inc. (Corning, NY, USA). BCA protein assay kit was obtained from Thermo Fisher Scientic. Chemiluminescent (CL) substrates were purchased from Ke Yue Zhong Kai Co., Ltd. (Beijing, China). The magnetic particles (MPs, 10 mg/ml) coated with streptavidin and suspended in solution were purchased from Beaver Beads Co., Ltd. Phosphate-buffered saline (PBS) buffer and bovine serum albumin (BSA) were from ZSGB-Bio (Beijing, China). The washing buffer was PBS containing 0.05% (v/v) Tween-20 (PBST). PBST containing 1% (w/v) BSA served as dilution buffer for HRP-labeled antibody, biotinylated antibody and standard series. The microplates were pre-coated with 300 µl 1% (w/v) BSA in PBS buffer at 4°C for 12 h and washed three times before use.