Immunohistochemical Staining of NSCLC Tissues

The standard streptavidin-biotin-peroxidase complex method (EnVision™ Detection System; Denmark) was used to perform immunohistochemical staining [60 (link)–63 (link)]. NSCLC tissues were cut into 4-mm sized sections, xylene was utilized to deparaffinize them, and graded ethanol series was used for rehydration. To stain them, they were treated with anti-PAK4 (1:500; Cell Signaling Technology) or GRP78 (1:500; Abcam) and subjected to incubation at 4 °C overnight. Two skilled observers independently graded the degree of immunostaining. The intensity and frequency of staining were estimated as previously described [20 (link)]. The staining index scores ranged from 0 to 12.