Immunohistochemical Evaluation of RUNX3 Expression

Immunohistochemistry staining were performed as described previously [13] (link). The primary rabbit anti-RUNX3 antibody (1∶200) (Medical and Biological Laboratories, Nagoya, Japan), anti-VIII (1∶100), anti-PAP (1∶100) (Santa Cruz, CA, USA) and the biotinylated anti-mouse IgG (1∶200) (Boster Biotech, Wuhan, China) were used. For the TMA staining evaluation, the immunoreactivity was assessed blindly by two independent observers using light microscopy (Olympus BX-51 light microscope), and the image was collected by Camedia Master C-3040 digital camera. The expression of RUNX3 was graded as positive when 10% of tumor cells showed immunopositivity. Biopsies with 10% tumor cells showing immunostaining were considered negative [15] (link).