Immunohistochemical Staining of Tumor Tissues

Tissue arrays were obtained from Superbiochips Laboratories (Seoul, Korea) as described previously.^{48 (link)} Each slide contained section of tumor and normal tissues obtained from cancer patient by biopsy or surgical resection. The tissues incubated with primary antibody for 1 h were then treated with anti-mouse biotinylated antibody (Vector Laboratories, Burlingame, CA, USA) for 1 h. Color reaction was developed by incubation with diaminobenzidine solution (Sigma-Aldrich, St. Louis, MO, USA) followed by counter staining with hematoxylin. Parallel sections incubated with normal IgGs instead of primary antibodies were used as negative controls. The overall staining results were scored from 0 to 3 based on the intensity and positive rate of staining. Intensity of staining was categorized as 0, negative; 1, weak; 2, intermediate; 3, strong. Stained tissue arrays were reviewed by experienced pathologists.