Goat anti rabbit antibody coupled to alexafluor 488

HeLa cells were seeded in 24-wells plate at 100.000 cells/well on glass coverslips (VWR, #631-0150). Twenty-four hours later, HeLa cells were treated with 20 μM pyridostatin (Sigma-Aldrich; CAS number 1085412-37-8) for 4 h, and then washed with PBS and fixed with 2% paraformaldehyde in PBS at room temperature for 10 min, washed with PBS and permeabilized for 15 min at room temperature with 10 mM Tris–HCl pH 7.5, 120 mM KCl, 20 mM NaCl, 0.1% Triton-X 100. Then, cells were washed with PBS and incubated for about 1 h at 37 °C in blocking buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 2% BSA, 0.2% fish gelatin, 0.1% Triton-X 100) prior to incubation overnight at 4 °C with γH2AX (Phospho S139) antibody (Abcam, #81299) diluted at 0.7 µg/mL in blocking buffer. Cells were then washed with 0.1% Tween20-PBS and incubated with secondary goat anti-rabbit antibody coupled to AlexaFluor 488 (Thermo Fisher Scientific) diluted at 2 µg/mL in blocking buffer for 1 h at room temperature. At last, cells were washed with 0.1% Tween20-PBS and stained with 0.1 μg/mL DAPI for 20 min at room temperature, and coverslips were mounted with Vectashield mounting medium (Vector Laboratories). Nuclear γH2AX foci staining overlapping with DAPI staining were quantified with ImageJ software. Quantifications of nuclear γH2AX foci induced by pyridostatin are represented normalized to non-treated (NT) conditions.