Immunohistochemical Analysis of Tumor Markers

Tissue sections (5 μm) of tumor xenograft or metastatic lung nodes were deparaffinized and blocked and incubated with primary antibody (Cell Signaling, Shanghai, China) against PEAK1 and Ki67 (tumor xenograft), E-cadherin and vimentin (metastatic lung nodes). Then, the slides were incubated with HRP-conjugated secondary antibody and peroxidase activity was visualized using a substrate solution of diaminobenzidine (DAB) containing 0.03% hydrogen peroxid, and counterstained with Harris hematoxylin. Tissue sections were washed in water, dehydrated, cleared, mounted and observed using Olympus FLUOVIEW Viewer software (Tokyo, Japan). Within the tissue core, the most representative tumor area of standardized size was selected at ×200 magnification. The staining intensity in this area was measured if the area contained >5% epithelial cells. Stains were scored as negative (−), weak (+), intermediate (++), and strongly positive (+++) as reported [14 (link)].