Anti brdu mouse igg

BrdU (10 280 879 001, Roche) was added to cell cultures at 20 μM for 1 h. Cells were fixed with 90% ethanol overnight at 4 °C, rinsed in PBS and incubated with 2 N HCl/0,5% Triton X-100 at RT for 30 min. After that, cells were suspended in 0.1 M sodium tetraborate for 2 min. Cells were incubated with anti-BrdU mouse IgG (M 0744, Dako) for 1 h at 37 °C, washed with PBS and incubated with AlexaFluor 488 anti-IgG Mouse (A-21202, Molecular Probes) for 1 h at RT. Cells were finally incubated with PBS containing 10 μg ml⁻¹ RNase A and 20 μg ml⁻¹ propidium iodide for 30 min at 37 °C and then analysed by flow cytometry on BD-FACSCanto II. The results were analysed with the FACSDiva 7.0 software.

For staining cells in S-phase, BrdU (10 280 879 001, Roche) was added to cell cultures at 20 μM for 1 h. For staining SSB foci undergoing repair, BrdU was added overnight. Cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100 and incubated with 40 U ml⁻¹ DNase I (M6101, Promega) and 20 U ml⁻¹ Exonuclease III (M1811, Promega) for 30 min at 37 °C. BrdU was revealed with anti-BrdU mouse IgG (M 0744, Dako) and Rhodamine Red-conjugated anti-mouse IgG (715-296-150, Jackson Immunoresearch laboratories).