Ab1838

Following micro-irradiation cells were fixed and permeabilized in 100% ice cold methanol for 15 min followed by 5 washes with PBS to allow cells to fully rehydrate. Prior to probing for the DNA strand break proteins cells were blocked in 1% BSA in PBS for 30 min at room temperature. Cells were then incubated with the primary antibodies for ɣH2AX (Millipore 05–363) and XRCC1 (abcam ab1838) diluted in 1% BSA in PBS at a 1:750 and a 1:50 respectively for 1 h at room temperature. For the cyclobutane pyrimidine dimer (CPD) immunofluorescence, cells were fixed and rehydrated as described, and then the DNA was denatured using 2N hydrochloric acid (HCl, Fisher #SA49) for 45 min at room temperature and washed 5 times with PBS. Samples are then neutralized in 50 mM Tris-HCl pH 8.8 (Amresco #J383) for 5 min at room temperature and washed 3 times with PBS. Samples are blocked in 5% normal goat serum (Pierce #31873) for 30 min at room temperature followed by an incubation with anti-CPD (Cosmo Bio clone TDM2) in 5% goat serum for 1 h at room temperature. All samples were then washed 3 times with PBS and incubated with Alexa-488 goat anti-mouse (ThermoFisher #A11034) in 1% BSA for 1 h at room temperature protected from light. Samples were washed 3 times with PBS and the nucleus was stained using NucBlue® fixed cell stain (DAPI, ThermoFisher #R37606) following the manufacturer’s instructions.