*2.2. Immunohistochemistry*

Tissue microarrays (TMAs) were constructed from paraffin blocks prepared from 425 NSCLC samples. Expression levels of β-catenin and NME1 proteins were analyzed using immunohistochemistry. In brief, serial sections of 4 μM in thickness were cut from TMA blocks, deparaffinized in xylene, and rehydrated through a series of decreasing concentrations of alcohols. Antigens were recovered by heating these sections in 10 mM (pH 6) citrate buffer for 10 min using a pressure cooker. These sections were then incubated with primary antibody β-catenin clone 17C2 (Leica Biosystems, Buffalo Grove, IL, USA) or NME1 clone 4B2 (GeneTex, Irvine, CA, USA) at 4 ◦C overnight. Immunoreactivity of each primary antibody was detected with Envision™ + peroxidase (Dako, Carpinteria, CA, USA). Antibody-bound peroxidase activity was visualized after incubating with chromogen 3,3 -diaminobenzidine (DAB) at room temperature for 1–5 min. Normal bronchial epithelial cells were used for positive control of staining, and primary antibody was replaced by immunoglobin for negative control. All sections were counterstained with Mayer's hematoxylin.
