Master c 3040

We performed immunohistochemistry according to the avidin-biotinylated HRP complex (ABC) method by using a standard ABC kit (Zhongshan biotech, Beijing, China). TMA slides was incubated with monoclonal Rabbit anti-Rap2B antibody (1:500) (Abcam, Cambridge, USA) overnight at 4 °C, while diaminobenzidine (DAB; Zhongshan Biotech, Beijing, China) was used to produce a brown precipitate. Then, a biotinylated secondary antibody (Zhongshan Biotech, Beijing, China) was incubated in the sections at room temperature for 30 min. The sections were followed by the incubation with streptavidin-peroxidase (Zhongshan Biotech, Beijing, China) for an additional 30 min. After rinsing with PBS three times for 5 min, the sections were stained using DAB (Zhongshan Biotech, Beijing, China) for 15 min, rinsed in distilled water, and counterstained with hematoxylin. The immunoreactivity was assessed blindly by two qualified pathologists using light microscopy (Olympus BX-51 light microscope), and the image was collected by Camedia Master C-3040 digital camera. The expression of Rap2B was graded as positive when >5 % of tumor cells showed immunopositivity. Biopsies with <5 % tumor cells showing immunostaining were considered negative.