Ab17798

Slides were deparaffinized and rehydrated in 10 mM PBS (pH 7.2). The tissue epitopes were unmasked using the Dako PT Link automated water bath heating process. The slides were incubated in Tris-EDTA retrieval solution (10 mM Tris, 1 mM EDTA pH 9.0) (ERCC1 and XPF staining) or citrate retrieval buffer of pH 6.0 (XPA staining) at 98 °C for 20 min. The slides were subsequently incubated for 1 h at room temperature (RT) with the primary mouse monoclonal antibody against ERCC1 (Dako, clone 4F9), mouse monoclonal antibody against XPF (Abcam [SPM228]: ab17798) or mouse monoclonal antibody against XPA (Abcam [12F5]: ab2352) diluted 1:100 in Dako REAL antibody diluent (Dako) and immunostained using anti-mouse/anti-rabbit immuno-peroxidase polymer (EnVision FLEX/HRP, Dako) at RT for 30 min, according to the manufacturer’s instructions. For visualization, the slides were treated with diaminobenzidine substrate-chromogen solution (DAB, Dako) for 5 min. Finally, the slides were counterstained with haematoxylin. Appendix or tonsil tissue samples were used as positive controls for ERCC1 or XPA and XPF, respectively. The same tissues with omitted primary antibodies served as a negative control.

The ChIP assays were performed according to the manufacturer’s protocol (Upstate Biotechnology, Inc., Lake Placid, NY), with the exception of the conditions for sonication that were changed to five times for 30 seconds each at 10% output. The antibodies used for the immunoprecipitations were anti-TRF2 (ab13579, abcam), anti-TRF1 (ab1423, abcam), anti-POT1 (ab21382, abcam), anti-HA (ab9110, abcam), anti-hist1H2AC (Novus Biologicals), anti-Histone H3.3 (ab62642, abcam) anti-γ-H2AX (pS-139) (ab2893, abcam), anti-ATM (pS-1981) (ab36810, abcam) and anti-XPF (ab17798, abcam). Non-specific rabbit polyclonal antibodies were used as a negative control. After reversing the protein-DNA cross-linking of the immunoprecipitated complexes, DNA was extracted for dot blotting analysis.