Cells were collected by centrifugation at 5000 × g for 5 min and washed once in PBS. Cells were then attached onto glass coverslips for 30 min at room temperature and fixed in cold (–20°C) methanol for 30 min. Coverslips were rehydrated in PBS for 10 min and blocked with 3% BSA in PBS for 30 min. Fixed cells were incubated with primary antibodies diluted in blocking buffer for 1 h. The following primary antibodies were used: FITC-conjugated anti-HA mAb (1:400 dilution, Sigma Aldrich, Clone HA-7), anti-Protein A pAb (1:400 dilution, Sigma Aldrich), anti-γH2A pAb (1:2000 dilution) (47 (link)) and anti-Rad51 pAb (1:2000 dilution) (48 (link)). After washing with PBS three times, cells were incubated with the Cy3-conjugated anti-rabbit IgG (1:400 dilution, Sigma Aldrich) for one hour at room temperature. After washing three times in PBS, coverslips were mounted with DAPI-containing VectaShield mounting medium (Vector Labs) and imaged using an inverted fluorescence microscope (Olympus IX71) equipped with a cooled CCD camera (Hamamatsu, Japan) and a PlanApo N 60× 1.42 NA oil lens. Images were acquired with the Slidebook5 software.