Immunohistochemical Analysis of SMOC2 and β-Catenin in CRC

Immunohistochemistry for SMOC2 and β-catenin and were performed on 4-μm TMA sections using a BOND-MAX automated immunostainer and a Bond Polymer Refine Detection kit (Leica Microsystems, Wetzlar, Germany) according to the manufacturer’s guidelines. The primary antibodies used were anti-SMOC2 (OriGene, Rockville, MD; 1:30, catalog number: TA351730) and anti-β-catenin (Novocastra Laboratories, Newcastle, UK; 17C2; 1:800). The expression of SMOC2 was determined by evaluating the whole field of each tumor core (4 mm in diameter). The intensity and percentage of tumor cells expressing SMOC2 in the cytoplasm were examined. Histoscores (H-scores) were measured by multiplying the intensity score (0 = negative; 1 = weak; 2 = moderate; 3 = strong) and percentage of positive tumor cells (range = 0–100), ranging from 0 to 300^{21 (link)}. For statistical analyses, we set a cutoff of 40 on the basis of the distribution of H-scores (mean value: 32.5). CRCs with H-score < 40 were classified as negative, while cases with H-score > 40 were classified as positive. Nuclear β-catenin staining was defined as positive when more than 10% of the cancer nuclei were stained for β-catenin. Immunohistochemical analysis was performed blinded to all other data.