Ab17993

MLH1, MPG, FEN1, POLβ and XRCC1 expression in primary tumours was examined from formalin-fixed paraffin-embedded samples. Tissues were sectioned at 4-μm and mounted on silanized slides, which were stored at 4°C. After deparaffinization and rehydration, the sections were quenched with 3% H₂O₂ in methanol to block endogenous peroxidase. Bovine serum albumin at 5% (BSA) was then applied to prevent non-specific binding. The sections were incubated with anti-MPG (dilution 1:100, Abcam, mouse, EPR10959(B)), anti-Polβ (dilution 1:500, Abcam, rabbit, ab26343), anti-FEN1 (dilution 1:800, Abcam, rabbit, ab17993), anti-XRCC1 (dilution 1:50, Abcam, mouse, ab1838), anti-MLH1 (dilution 1:100, Abcam, ab92312) and PCNA (dilution: 1:400, mouse, #2586) antibodies, and then incubated with appropriate secondary antibodies (DAKO). Diaminobenzidine (DAB) was used as chromogen and the sections were counterstained with haematoxylin. Omission of the primary antibody was used as a negative control. Protein expression was evaluated using Quick Score (QS) (by assessing both staining intensity and the percentage of the stained area with a given intensity) and dichotomized in low (score between 0 and 4) and high expression (score between 5 and 12) (Supplementary Material and Methods). Only stained nuclei of malignant cells were assessed.