Ab133311

Total protein was extracted from the tissue samples and cells, and the protein concentrations were quantified using a BCA kit (Yeasen, Shanghai, China). Equivalent proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Then, the membranes were blocked and incubated with primary and secondary antibodies. We used ECL chemiluminescence to detect protein signals. GAPDH was used as the internal reference protein. Antibodies against the following proteins were used: GAPDH (60004-1-Ig, Proteintech), FEN1 (ab133311, Abcam), Cdc25C (ab32444, Abcam), CDK1 (ab133327, Abcam) and Cyclin B1 (ab32053, Abcam).

The sections were baked at 56°C for 2 h for dewaxing, boiled in citrate buffer for antigen retrieval, and blocked using 3% hydrogen peroxide. The contents were incubated with the primary antibody against FEN1 (1: 200, ab133311, Abcam) at 4°C overnight and biotinylated with a goat anti-rabbit secondary antibody for 1 h. At last, the reaction was visualized using DAB, and the sections were counterstained with hematoxylin. IHC scores were calculated by multiplying the percentage and intensity score of stained cells (staining intensity: negative = 0, weak = 1, moderate = 2, strong = 3, and staining extent: 0 = no staining, 1 = 0%-25%, 2 = 25%-50%, 3 = 50%-75% and 4 = 75%-100%). The total score was calculated as intensity score × extent score. Scores of > 4 were regarded as having high FEN1 expression while those with 1, 2, 3 and 4 were regarded as having low FEN1 expression.