Autostainer instrument

For immunofluorescence, cells were seeded in chamber slides (Nunc) and allowed to plate for 72–96 h. Cells were then treated with or without oxaliplatin (Fresenius Kabi) at a final concentration at 8 μM for 24 h (see supplementary note 2). Staining was performed using an Autostainer instrument (Dako). Briefly, cells were permeabilized by incubation with PBS containing 0.25% Triton X-100 (10 min) and thereafter blocked with 10% goat serum in PBS for 1 hour. Slides were treated with peroxidase block (5 min), and then incubated with a polyclonal anti-γ-H2AX rabbit antibody (dilution 1:1200) for 20 min to detect DNA double strand breaks. Signal was generated by use of goat anti-rabbit secondary antibody coupled to HRP (20 min) (Dako) with a Texas Red-based fluorescent tyramide substrate (3 min) (Dako). To terminate signal generation, slides were treated with peroxidase block. Slides were subsequently incubated with anti-ERCC1 antibody 4F9 at 1.66 μgml⁻¹ (corresponding to a dilution of 1:300) for 20 min. Antibody 4F9 was visualized by use of goat anti-mouse secondary antibody coupled to HRP with a FITC-based fluorescent tyramide substrate (3 min). Finally, slides were washed and counterstained with DAPI and mounted.

For immunostaining of paraffin embedded cell lines (see supplementary note 2) and FFPE patient specimens, all reagents came from the EnVision™ FLEX, High pH kit (K8010) (Dako). Antigen retrieval was performed at pH 9 for 20 min using a PT Link module (Dako). Staining was performed in an Autostainer instrument (Dako) or manually according to manufacturer's instructions. Briefly, slides were treated with peroxidase block (5 min), then incubated with anti-ERCC1 antibody 4F9 at 1.66 μgml⁻¹ for 20 min. Visualization of antibody binding was done by incubation with a labeled HRP-polymer for 20 min and signal was generated with a 3.3′-diaminabenzidin (DAB) chromogen (10 min). Hematoxylin was used for counterstaining.