Duolink detection reagents red

PLA was performed using Duolink PLA Technology (Merck). Cells were incubated 10 min with 25μM EdU before HU treatment (3mM, 4h). Samples were incubated with 0.1% formaldehyde in PBS for 5 min, and pre-extracted in CSK buffer (10 mM Pipes pH 7, 0.1 M NaCl, 0.3 M sucrose and 3 mM MgCl₂), prior to fixation. Click Reaction (100 mM Tris-HCl pH 8, 100 mM CuSO₄, 20 mg/mL sodium-L-ascorbate and 10mM azide-biotin) was performed according to the manufacturer’s guidelines for 1-2h at 37°C. Duolink Blocking Solution was replaced by 5% BSA, 10% Donkey serum in PBS. First and secondary antibody binding, ligation and amplification reactions were performed according to the manufacturer’s guidelines. Duolink in situ PLA probe anti-rabbit PLUS, Duolink in situ PLA probe anti-mouse MINUS and Duolink-Detection Reagents Red (Merck) were used to perform the PLA reaction. Finally, nuclei were stained with DAPI and mounted in ProLong Gold AntiFade reagent (Invitrogen). Antibodies were used at 1:500 dilution. PLA foci were automatically quantified using Metamorph v7.5.1.0 software (Molecular Probes).