Lsm510 laser scanning confocal microscope

HeLa cells were grown on culture slides for two days until the density increased to 50% at the time of treatment. Following the aforementioned treatments, cells were washed in PBS, fixed in 4% paraformaldehyde for 30 min, permeabilized in 0.5% Triton X-100/PBS for 15 min and blocked in blocking buffer [1% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany)] in PBS) for 30 min. Immunostaining was performed using an anti-phosphorylated histone H2AX (γH2AX; Ser¹³⁹) antibody (mouse anti-human, monoclonal antibody, catalog no. 05-636, EMD Millipore, Billerica, MA, USA, at a dilution of 1:500) for 2 h at room temperature in a humidified chamber. Following three 10-min washes, the cells were incubated with the goat anti-mouse Alexa Fluor 568-conjugated secondary antibody (Invitrogen; Thermo Fisher Scientific, Inc.; catalog no. A11004, 1:1,000 dilution). The DNA was stained using a mounting medium with DAPI (Sigma-Aldrich; Merck KGaA). Immunofluorescence microscopic imaging was performed using a LSM 510 laser-scanning confocal microscope (Zeiss GmbH, Jena, Germany). All experiments were performed at least three independent times.