Anti rabbit alexafluor 488 antibody

Cells were seeded onto 96-well cell culture plates and allowed to attach overnight before exposure to treatments. Following treatment, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, blocked with 5% BSA, and subsequently incubated with primary antibodies (anti-phospho-histone H3 Ser10, Abcam, ab5176, 1:200; anti-γH2AX, CST, 80312, 1:200) overnight at 4 °C. This was followed by incubation with secondary antibodies (anti-Rabbit AlexaFluor 488 antibody, Jackson Immunoresearch, 711-545-152, 1:200; anti-Mouse AlexaFluor 594 antibody, Jackson Immunoresearch, 715-585-151, 1:150, 1:200) for 1 h at RT. Nuclei were counterstained with DAPI (1 mg/mL, Servicebio, G1012). Cells were observed and imaged under the ImageXpress Micro Confocal High-Content Analysis System (Molecular Devices). Cell nuclei and positive cells were quantified using ImageJ software (version 1.53k). Information on antibody dilution ratios, manufacturers, and catalog numbers can be found in Supplementary Table 7.

Briefly, cells were labeled with 10 μM BrdU (Sigma-Aldrich, CAS:59-14-3) for 48 h before being subjected to the indicated treatments (in the absence of BrdU). Cells were then fixed with 4% polyformaldehyde for 15 min. BrdU detection under non-denaturing conditions was performed using a specific rat monoclonal antibody (anti-BrdU [clone BU1/75 (ICR1)], Abcam, ab6326, 1:200), while γH2AX was detected using rabbit anti-γH2AX antibodies (ABclonal, AP0687, 1:200) overnight at 4 °C. After rinsing, cells were incubated with anti-Rat AlexaFluor 594 antibody (Jackson Immunoresearch, 712-585-153, 1:200) and anti-Rabbit AlexaFluor 488 antibody (Jackson Immunoresearch, 711-545-152, 1:200) for 1 h at RT. Finally, cells were stained with DAPI and imaged using an ImageXpress Micro Confocal High-Content Analysis System (Molecular Devices). Cell nuclei and positive cells were quantified using ImageJ software (version 1.53k). Information on antibody dilution ratios, manufacturers, and catalog numbers can be found in Supplementary Table 7.