Pc130

Meiotic spreads were performed as previously described[53 (link)]. Primary antibodies used in this study: rabbit anti-SYCP3 (1:500, Abcam ab13840); mouse anti-γH2AX (1:500, Millipore JBW301); mouse anti-SYCP3 (1:500 Abcam ab20244); rabbit anti-RAD51 (1:250, Millipore PC130).

Approximately 5 x 10⁴ mouse ES cells were seeded on poly-D-Lysine coated coverslips (neuvitro GG-12) and cells were irradiated (IR) with 10Gy of γ-radiation after 24 hours. After 3 hours post IR, the cells were treated with hypotonic solution (85.5 mM NaCl, 5mM MgCl2, pH 7) for 10 min followed by fixing solution (4% Paraformaldehyde, 10% SDS in PBS) for 10min. Cells were incubated overnight at 4°C with primary antibodies: γH2AX (1:500, Millipore JBW301), RAD51 (1:250, Millipore PC130) diluted in antibody dilution buffer (1% BSA, 0.3% TritonX100, 5% goat serum in PBS). The following day, cells were washed three times with PBS containing 0.2% Triton X 100 (PBST) and incubated with secondary antibodies (Alexa-fluor anti-mouse 594 [Invitrogen A11005], and anti-rabbit 488 [Invitrogen A11034]; 1:500) diluted in PBS at 37°C for 1 h. The cells were washed three times with PBST and stained for DAPI (1:50,000, Sigma 11190301) for 5 min. The coverslips were mounted on clean labeled slides with anti-fade mount (Invitrogen P36930) and imaged in Zeiss 710 confocal microscope (63X oil immersion objective) and analyzed utilizing Carl Zeiss zenblue 2.6 software.

Meiotic spreads were prepared from the testes of 4–6-week-old male mice. The testes were briefly rinsed in PBS and then transferred to hypo-extraction buffer (30 mM Tris pH 8.2, 50 mM sucrose, 17 mM citric acid, 5 mM EDTA, 0.5 mM DTT, 0.1 mM PMSF). Gently the tunica was peeled off the testes and the tubules were teased out using fine forceps and incubated in this solution for 30 min at room temperature. On a clean labeled (prerinsed with PFA, pH 9.2 adjusted with 50 mM boric acid) slide, 25 μl of 0.1 M sucrose solution was placed and a small portion of digested tissue section from the previous step was placed. Using a fine needle this tissue was shredded and spread on the slide. These slides were kept overnight in a humid chamber to dry slowly. After the slides were ready the immunofluorescence staining was performed as above. Primary antibodies used in the study: rabbit anti-SCP3 (1:500, Abcam ab15093); mouse anti-γH2AX (1:500, Millipore JBW301); mouse anti-SCP3 (1:500 Santa Cruz sc-74568); rabbit anti-RAD51 (1:250, Millipore PC130). Secondary antibody staining was performed as described above in Immunofluorescence section.

Cells were seeded onto slides and transfected with empty vector, Flag-SMRAD51, Flag-WTRAD51, Flag-RAD51 K133A and Flag-RAD51 K133R, HA-WTRAD51 or HA-RAD51-T131P. Forty-eight hours after transfection, the cells were washed with PBS, treated with CSK buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl₂, 10 mM Pipes pH 6.8, 1 mM EGTA, 0.2× Triton and protease inhibitor cocktail (Complete ULTRA Tablets, Roche, Basel, Switzerland) and fixed in 2% paraformaldehyde for 15 min. The cells were then permeabilized in 0.5% Triton-X 100 for 10 min, saturated with 2% BSA and 0.05% Tween 20 and probed with anti-Flag (1/400, F3165 Sigma-Aldrich, St Louis, MO, USA), anti-HA (1/100, sc-7392, Santa Cruz, Dallas TX, USA), or anti-RAD51 (1/500, PC130, Millipore, Burlington, MS, USA) antibodies for 2 h at RT or overnight at 4°C. After three washes in PBS-Tween 20 (0.05%) at RT, the cells were probed with Alexa-coupled anti-mouse or anti-rabbit secondary antibody (1/1000, Invitrogen Life technologies, Waltham, MA, USA) for 1 h at RT. After three washes, the cells were mounted in DAKO (Agilent, Santa Clara, CA, USA) mounting medium containing 300 μM DAPI and visualized using a fluorescence microscope (Zeiss Axio Observer Z1) equipped with an ORCA-ER camera (Hamamatsu). Image processing and focus counting were performed using ImageJ software.

MEFs were generated from E13.5 embryos and cultured in 10% FBS at 5% O₂. Cell growth analyses were done by seeding 10cm plates with 5 X 10⁵ cells and counting the cells every day. Metaphases were collected after a 24 hour treatment with 100nM MMC and spread as previously described [52 (link)]. Immunofluorescence of RAD51 foci was done by seeding cells on coverslips, irradiating with 1000 rads the following day. Fixed cells were collected after 3 hours and cells were stained with Mouse anti-γH2AX (1:500, Millipore JBW301) and rabbit anti-RAD51 (1:250, Millipore PC130).

Cells were plated on poly-d-lysine coated coverslips (50 μg/ml) (Sigma Aldrich) 24 h prior to treatment with ionising radiation. Coverslips were placed into ice-cold pre-extraction buffer for 7 min (10 mM PIPES pH 6.8, 300 mM sucrose, 20 mM NaCl, 3 mM MgCl₂ 0.5% Triton-X 100), then fixed for 10 min in 3.6% PFA. After washing in phosphate-buffered saline (PBS) and 1 h block (10% FCS in PBS), cells were incubated in primary antibody overnight at 4 °C and secondary antibody at 1:1000 for 1 h at room temperature. Cells were washed three times with PBS and mounted onto glass slides with Prolong gold anti-fade reagent with DAPI (Life Technologies). Staining was assessed through eyepiece counting using a Zeiss Axiovision 3 immunofluorescent microscope and representative images were taken using ZEN Blue software (v2.3). A minimum of 50–150 cells was counted for each experimental repeat. Antibodies used include: γH2AX (Millipore: 05-636; 1:500), 53BP1 (Novus: NB100-304; 1:300), RAD51 (Millipore: PC130; 1:500), RPA (Millipore: NA18; 1:200), CENPF (CST: 58982; 1:1000) and mitosin (BD: 610768; 1:1000).