Donkey anti rabbit igg fitc secondary antibody

S180 cells were pretreated with 10 μM genistein for 24 h, followed by exposure to 2 Gy X-rays. At 2 h and 12 h post irradiation, the cell suspensions were collected and fixed (methanol:acetone = 3:1), then dripped onto glass slides and allowed to dry. The fixed cells were permeabilized with 0.1% Triton X-100 and subsequently blocked with 5% BSA at room temperature for 1 h. The cells were then incubated overnight at 4°C with primary monoclonal antibodies against Ku70/80 (mouse monoclonal to Ku70/80), Rad51 (rabbit monoclonal to Rad51) and DNA-PKcs (rabbit monoclonal to DNA-PKcs) (Abcam, Cambridge, USA, 1:200 dilution). After primary antibody incubation, the cells were washed with PBST and incubated with donkey anti-mouse IgG-R secondary antibody and donkey anti-rabbit IgG-FITC secondary antibody (Santa Cruz, Dallas, USA, 1:500 dilution) for 1 h at room temperature. Nuclei were counterstained with mounting medium DAPI (1.5 μg/ml, VECTASHIELD Mounting Medium, Vector Lab. Inc., Burlingame, USA). Finally, Ku70/80, DNA-PKcs and Rad51 foci were detected with a laser scanning confocal microscope (LSM 700, Zeiss, Oberkochen, Germany). Mean values of foci were determined by 50 cells.