Sc 130867

Human tissue specimens (HBre-Duc140Sur-01) was purchased from Shanghai Outdo Biotech CO. The primary antibodies against Twist (Merck Millipore, ABD29) or ROR1 (Santa cruz, sc-130867) were diluted 1:3000 or 1:800, respectively, and then incubated at 4 °C overnight in a humidified container. Following washing three times with PBS, the section was treated with a non-biotin horseradish peroxidase detection system according to the manufacturer's instructions (Dako). The tissue samples were examined by two pathologists who were blinded to the pathological information, and immunoreactivity. For Twist and ROR1, each tissue sample was scored according to its staining intensity (0, none; 1, weak; 2, moderate; 3, strong), and the percentage of stained cells (0, 0%; 1, 1-24%; 2, 25-49%; 3, 50-74%; 4, 75-100%). The score for each tissue was calculated by multiplying the staining intensity and the percentage of stained cells, yielding a value between 0 and 12. Then, the ROC curve analysis was used to determine the immunohistochemical cut-off for high or low expression. Cancers with scores above the cut-off value were considered to have high expression of the indicated molecule and vice versa.