Immunohistochemistry

Immunohistochemical analysis was performed on formalin-fixed paraffin-embedded section. All tissue samples from RC, DRM, and PRM were fixed in 10 % buffered formalin and embedded in paraffin. We prepared 4-μm-thick serial section which were deparaffinized in xylene, rehydrated in graded ethanol, and washed with phosphate-buffered saline. Endogenous peroxidase was inhibited with 3 % hydrogen peroxide. Tissue sections were incubated for 30 min with the anti-CD105 primary monoclonal antibody (mouse anti-human, clone SN6h, Dako Corporation, Denmark) at a 1:10 dilution. Primary antibody binding site was visualized using a secondary antibody detection kit (Envision + kit; Dako, Denmark).

The staining was visualized with diaminobenzidine (DAB). Tissue sections were counterstained with hematoxylin. Brown staining for CD105 was considered positive. Distant normal mucosa free of tumor were used as positive controls, and the primary antibody was replaced with phosphate-buffered saline solution for negative controls.