Immunohistochemistry was performed on 4-μm sections of paraffin-embedded tissue specimens of surgical margins obtained from 235 patients with OSCC. Axin2 (Rabbit polyclonal IgG, working dilution 1:500; ab32197, Abcam, Cambridge, UK) and Snail antibodies (Rabbit polyclonal IgG, working dilution 1:1000) were used as primary antibodies. The Snail antibody was prepared as previously described (25 (link)). Endogenous peroxidase activity was inhibited using a mixture of H2O2 and methanol (1:40), and antigen retrieval was performed by the pressure-cooking method using citrate buffer (pH 6.0, Sigma-Aldrich, Darmstadt, Germany) for the deparaffinized tissue sections. The Real Envision HRP Rabbit/Mouse detection system (Dako, Glostrup, Denmark) was used as a secondary antibody, and immunoreactivity of the tissue sections against Axin2 and Snail was visualized using 3,3′-diaminobenzidine.
The histoscore for the immunoreactivity of both Axin2 and Snail in the surgical margins of OSCC was calculated according to the staining intensity and percentage of positive cells using the weighted histoscore method (26 (link)). Patients were subdivided into two groups according to the histoscore: low (histoscore 0–100) and high (histoscore 101–300) expression groups.
The histoscore for the immunoreactivity of both Axin2 and Snail in the surgical margins of OSCC was calculated according to the staining intensity and percentage of positive cells using the weighted histoscore method (26 (link)). Patients were subdivided into two groups according to the histoscore: low (histoscore 0–100) and high (histoscore 101–300) expression groups.