Alexa 488 or 555 conjugated secondary antibodies

In this study, EdU was detected in all immunofluorescence experiments to allow identification of S phase cells. Cells were treated with 10 μM EdU for 30 min before fixation to label the S phase cells. At each time point after IR, the EdU-treated cells were washed once with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS for 10 min at 4 °C, followed by permeabilization with 0.5% Triton X-100 in PBS for 5 min at 4 °C. Primary antibodies were then applied for 30 min at 37 °C, followed by reaction with Alexa 488- or 555-conjugated secondary antibodies (Life Technologies, USA) for 30 min at 37 °C. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). All of the antibodies were diluted in 5% skim milk/Tris-buffered saline with 0.1% Tween 20.
After immunofluorescence staining, EdU was detected following the manufacturer’s protocol. Images were acquired using a fluorescence microscope (DM6000B, Leica, Germany) or a confocal fluorescence microscope (LSM800, Zeiss, Germany). For image acquisition using DM6000B (Leica), x63 or x100 objective lenses, CCD camera (Orca R2, Hamamatsu Photonics), and FW4000 software were used. For image acquisition using LSM800 (Zeiss), x63 or x100 objective lenses, GaAsP detector, and ZEN 2.1 image acquisition software were used.

To analyze BRCA1, RPA, and 53BP1 foci formation, U2OS cells were grown on glass cover slips and either pre-extracted (for BRCA1 foci analysis) with extraction buffer (3 mM MgCl₂, 0.3 M sucrose, 25 mM HEPES, 25 mM NaCl, 25 mM EDTA, and 0.5% Triton X-100) for 5 min at room temperature, and then fixed with 4% PFA for 15 min, or fixed directly with PFA without pre-extraction. Fixed cells were washed with PBS, permeabilized with 0.5% Triton X-100 for 20 min, and then blocked with 10% NBS in PBS for 30 min. Primary antibody incubations were for 1 h at room temperature, except for the RPA antibody, which was incubated at 37 °C. Antibodies and dilution ratios used in this study were: RPA2 (1:200) (Novus Biologicals, Littleton, CO, USA), BRCA1 D-9 (1:200) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and 53BP1 (1:500) (Novus Biologicals). Cells were washed 3 times in PBS prior to incubation with either Alexa 488 or 555-conjugated secondary antibodies (Life Technologies/Invitrogen, Grand Island, NY, USA) diluted in 10% NBS (1:500) for 1 h at room temperature. This was followed by 3 more washes in PBS, and then cells were mounted onto glass slides with Prolong Gold Antifade reagent with DAPI (Invitrogen/Life Technologies).