Dapi fluroshield

Cells were seeded onto Lab-Tek II eight-well chamber slides (Thermo Fisher Scientific) and treated with the appropriate agents before washing in warm PBS. Cells were then fixed using 4% Paraformaldehyde (Life Technologies 28906) for 10 min at room temperature. Fixed cells were then washed in PBS and permeabilized in 0.1% Triton-X. Cells were washed in PBS prior to blocking for 1 h at room temperature with shaking in blocking buffer (0.1% Tween-20 + 5% FCS) then incubated with the appropriate primary antibody (DNMT1 (SigmaAldrich D4567) used 1:100, BRCA1 phospho S1423 (Abcam ab47325) used at 1 μg/ml and γH2A.X [3F2] (Abcam ab22551) used at 3 μg/ml) overnight at 4°C. After washing each chamber in blocking buffer, the corresponding secondary antibody was added, the chamber slide wrapped in foil and incubated at room temperature for 1 h. Each well was then washed with PBS. The chamber apparatus was removed and DAPI/Fluroshield (Abcam ab104139) was added to the slide. After coverslipping and sealing, slides were stored in the dark at 4°C prior to imaging. Images were captured on the Andor Spinning Disk Confocal Microscope. FITC was captured at an exposure time of 200 ms, Alexa Fluor at 100 ms and DAPI at 100 ms.