γ-H2AX staining

Sections were deparaffinised in xylene and rehydrated through a graded series of 100 – 50% alcohol solutions, followed by immersion in MilliQ water. Antigen retrieval was performed using 0.01 M citrate buffer, pH 6.0. Sections were blocked in a 4% (w/v) solution of BSA in 1x TBS-T (0.01 M Tris, 150 mM NaCl, and 0.1% (v/v) Tween-20) and then incubated with 1:250 dilution of rabbit anti-γH2AX (Cell Signalling) in blocking buffer overnight at 4°C. After rinsing in 1x TBS-T, primary antibodies were detected with a 10 μg/ml solution of Alexa Fluor 488-conjugated goat anti-rabbit IgG in blocking buffer for 2 h. This solution also contained a 1:1000 dilution of the DNA counterstain DRAQ5 (Abcam). Finally, sections were mounted with Mowiol mounting medium (0.1 M Tris, pH 8.5, 10% (w/v) Mowiol 4-88, 25% (v/v) glycerol and 10 μg/ml 1,4-diazabicyclo(2,2,2)octane (DABCO)). Images were captured by confocal microscopy using the Leica S5 microscope equipped with a 40x PlanApochromat objective. Images were rendered in ImageJ.