P 4e bpl

Tumor tissue derived from surgical specimens was fixed in 7.5% formalin, and IHC was performed on 4-mm paraffin sections from one representative tissue block per patient following the manufacturer’s instructions. The sections were stained with a monoclonal antibody (mAb) against EGFR, phosphorylated- (p) mTOR, p-4EBPl, and p-S6K1 (1:100 dilution, Cell Signaling [Boston, Massachusetts, USA]) and counterstained with hematoxylin. Protein expression was detected using horseradish peroxidase- (HRP) conjugated rabbit anti-mouse immunoglobulin G (IgG) secondary antibody, followed by colorimetric detection using Diaminobenzidine (DAB). The positive expression was determined under a microscope (×400) by the presence of clear brown granules in the membrane or cytoplasm and/or nucleus. Five fields were selected randomly, and a total of 200 cells were counted. The intensity of staining was scored “−” for colorless, “+” for light yellow, and “++” for yellow and brown–yellow. The positive index scored negative (−) when the number of positive cells was less than 10%, weakly positive (+) when the number of positive cells was 10%–50%, and strongly positive (++) when the number of positive cells was more than 50%.