Immunohistochemical staining was performed on formalin-fixed Paraffin sections. Briefly, the sections were deparaffinised in xylene, rehydrated with graded ethanols, autoclaved for antigen repair and treated with 3% hydrogen peroxide solution to inactivate the endogenous peroxidase. After blocking for 30 min in 10% fetal calf serum and rinsed in PBS, the sections were incubated overnight at 4°C with the first antibody, such as rabbit anti-human KDF1 antibody (cat.no. PA5-55926, Invitrogen, MA, USA, dilution 1:200) and rabbit anti-human ki-67 antibody (cat.no. 790-4286, Roche, AZ, USA, dilution 1:2). Then, the sections were washed three times, incubated with the second antibody for 30 min, washed again and developed with diaminobenzidine. Finally, each section was counterstained with haematoxylin. Normal homologous serum was used to replace the first antibody as a negative control. According to the immunostaining intensity, the level of KDF1 was scored by two experienced pathologists in a blind manner: 0, negative; 1, weak; 2, medium; 3, strong. The slides with different score obtained by the two pathologists were reviewed again until the agreed score was made.

For the evaluation of the ratio of ki-67 positive cells in the tumor tissues, at least 15 pictures were taken from each section. The number of ki-67 positive nucleus and total nucleus in each picture were counted and the ratio of ki-67 positive nucleus was calculated. The average value of all the pictures from a section was used as the ki-67 positive ratio of the section.

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