Ab1423

Cells grown on glass coverslips were fixed in 4% paraformaldehyde/PBS for 15 min and then permeabilized with 0.1% Triton-X 100/PBS at 37°C for 30 min. Then, the cells were finally blocked with 5% goat serum/PBS at 37°C for 3 h. Immunofluorescence was performed using standard methods, and the slides were incubated alternately with BG4 (40 ng/μl), anti-FLAG antibody (#2368, Cell Signaling Technology), γH2AX antibody (#9718, Cell Signaling Technology; ab26350, Abcam), TRF2 antibody (ab13579, Abcam), TRF1 antibody (ab1423, Abcam) or POT1 antibody (ab21382, Abcam) at 37°C for 3 h. The glass coverslips were washed six times with blocking buffer and were then incubated with the anti-rabbit Alexa 488-conjugated antibody (A21206, Life Technology), the anti-mouse Alexa 555-conjugated antibody (A21427, Life Technology), and 0.5 μg/ml of DAPI (Invitrogen) at 37°C for 3 h. The glass coverslips were again washed six times with blocking buffer, and then, digital images were recorded using a LSM710 microscope (Zeiss, GER) and analyzed with ZEN software. Fifty nuclei were counted in each group.