4.12. Detection of phospho-53bp1 foci by Immunofluorescence Microscopy

Cells were seeded onto poly-lysine treated glass coverslips in 24 well plates and incubated overnight. Anoxic samples were transferred to the anaerobic chamber and equilibrated for 2 h. Cells were incubated for 4 h in the presence or absence of TPZ under aerobic and anoxic conditions. Following drug treatment, media was replaced with fresh drug-free media and the cells incubated for 1 h under aerobic conditions. After a 1 h incubation cells were washed in PBS (3×) and fixed in cold PBS/2% paraformaldehyde for 20 min at room temperature. Cells were washed with PBS/1% BSA (3×) and permeabilized in PBS/1% BSA/0.2% Triton-X100 for 10 min. After 3 washes with PBS/1% BSA a blocking buffer (PBS/1% BSA/5% normal goat serum) was applied for 30 min before addition of the primary antibody, rabbit phospho-53BP1 Ser1778 (Cell Signaling Technologies, Danvers, MA, USA) in PBS/1% BSA/1% normal goat serum. Cells were incubated in primary antibody at 4 °C overnight, washed in PBS/1% BSA (3×) and incubated with secondary antibody (goat anti-rabbit alexafluor 488; Invitrogen) for 2 h at room temperature. The nucleus was stained with DAPI (Invitrogen, Carlsbad, CA, USA) and cells washed 4 times with PBS prior to mounting on microscope slides using prolong gold anti-fade reagent (Invitrogen). Images were taken on a DMR microscope (Leica, Wetzlar, Germany).