RAD51 immunofluorescence assay

The pre-treatment and post-IC tumour blocks were stained using identical procotols previously validated at our institution (Graeser et al, 2010). Irradiated FFPE mouse xenograft was used as a positive control. Immunofluorescence analysis was carried out on 3 μm sections of FFPE tumour material. Following antigen retrieval by microwaving at pH 9 (DAKO pH 9 buffer) for 18 min, followed by 20 min cooling in buffer, sections were treated with Triton 0.2% for permeabilization for 20 min, washed in phosphate-buffered saline (PBS), treated with 100 μl of DNAse I (Roche) 1 : 10 000 for 1 h at 37 °C and blocked with immunofluorescence buffer (IFF; 1% bovine serum albumin, 2% fetal bovine serum; PAA gold in PBS) for 30 min at room temperature. Sections were stained with geminin antibody (10802-1-AP, ProteinTech Group). 1 : 400 in IFF for 1 h at room temperature, washed with PBS, followed by anti-rabbit Alexa488 (Invitrogen, #A21121) conjugate 1 : 1000 in IFF for 1 h at room temperature, washed with PBS, fixed with 4% paraformaldehyde (PFA) solution for 15 min, stained with RAD51 antibody (Clone 14B4; GeneTex) 1 : 100 in IFF for 1 h at room temperature, washed with PBS, followed by anti-mouse Alexa555 conjugate (Invitrogen, #A21429) 1 : 1000 in IFF for 1 h at room temperature, washed in PBS with 4', 6 diamidino 2 phenylindole (DAPI; 1 : 10000) for 15 min, and sections were fixed again with 4% paraformaldehyde (Sigma-Aldrich). The protocol for γH2AX staining was similar, with primary antibody 1 : 500 (phospho- Histone H2AX Ser139 Clone JBW301; Millipore) in IFF for 1 h at RT.