Mouse anti idu antibody

To detect nascent ssDNA, cells were labelled for 20 min with 50 µM IdU (Sigma-Aldrich), immediately prior the end of the indicated treatments. To detect parental ssDNA, cells were labelled for 24 h with 50 µM IdU (Sigma-Aldrich), released in a fresh DMEM for 2 h, then treated as indicated. For immunofluorescence, cells were washed with PBS, permeabilized with 0.5% Triton X-100 for 10 min at 4 °C and fixed in 3% PFA/2% sucrose. Fixed cells were then incubated with mouse anti-IdU antibody (Becton Dickinson) for 1 h at 37 °C in 1% BSA/PBS, followed by species-specific fluorescein-conjugated secondary antibodies (Alexa Fluor 488 Goat Anti-Mouse IgG (H + L), highly cross-adsorbed—Life Technologies). Slides were analysed with Eclipse 80i Nikon Fluorescence Microscope, equipped with a Video Confocal (ViCo) system. For each time point, at least 100 nuclei were examined by two independent investigators and foci were scored at 60×. Quantification was carried out using the ImageJ software. Only nuclei showing >10 bright foci were counted as positive. Parallel samples either incubated with the appropriate normal serum or only with the secondary antibody confirmed that the observed fluorescence pattern was not attributable to artefacts.

To detect nascent single-stranded DNA (ssDNA), cells were labelled for 30 min with 250 μM IdU (Sigma-Aldrich), immediately prior to the end of the CPT treatments. For immunofluorescence, cells were washed with PBS, permeabilized with 0.5% Triton X-100 for 10 min at 4°C, and fixed as previously described (22 (link)). Fixed cells were then incubated with mouse anti-IdU antibody (Becton Dickinson) for 1 h at 37°C in 1% BSA/PBS, followed by incubation with species-specific fluorescein-conjugated secondary antibodies (Alexa Fluor 488 Goat Anti-Mouse IgG (H+L), highly cross-adsorbed; Life Technologies). Slides were analysed with an Eclipse 80i Nikon Fluorescence Microscope, equipped with a VideoConfocal (ViCo) system. For each time point, at least 100 nuclei were examined by two independent investigators and foci were scored at 60×. Quantification was carried out using the ImageJ software. Only nuclei showing more than 10 bright foci were counted as positive. The results for parallel samples incubated either with the appropriate normal serum or only with the secondary antibody confirmed that the fluorescence pattern observed was not attributable to artefacts.