Immunohistochemistry assay were performed on tissues collected from 68 pathologically confirmed patients with RCC and 6 normal renal tissues. Immunohistochemistry experiments were performed as described previously (11 (link)). In brief, representative samples were placed into 4% paraformaldehyde overnight and then 5-mm paraffin sections were prepared for the experiments. Sections were deparaffinized in xylene, rehydrated which was hydrate by placing in 95, 70, 50 and 30% ethanol for 2 min each, and endogenous peroxidase activity was quenched by 3% hydrogen peroxide in methanol. The sections were submerged in 10 mM citrate buffer (pH 6.0) and microwaved for 8–15 min for antigen retrieval. Non-specific binding was blocked by incubation with normal goat serum (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) for 1 h at room temperature, and the slides were incubated with NEDD9 rabbit monoclonal primary antibodies (cat. no. ab37161; 1:200; Abcam, Cambridge, MA, USA) at 4°C overnight. After washing, sections were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG at room temperature for 1 h (cat. no. SPN-9001; 1:50; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.). Color was developed with the DAB Horseradish Peroxidase Color Development kit and imaged using bright field light microscopy.