Tissue microarray (TMA) and immunohistochemistry (IHC)

TMAs were purchased from BioMax (USA). Sections were arranged in duplicate cores per case. TMAs were treated with xylene, then 100% ethanol, and then decreasing concentrations of ethanol. After antigen retrieval, they were blocked and stained with antibodies against ezrin or cortactin, followed by secondary antibody incubation and the standard avidin biotinylated peroxidase complex method. Hematoxylin was used for counterstaining and images were taken with an upright microscope Nikon system (Nikon, JAPAN).

A modified Histo-score was used to perform IHC scoring, which was assessed by both intensity of staining and percentage of positive area. The extent of the positive area was scored on a scale of 0–4 as follows: 0, 0%; 1, 1–10%; 2, 11–50%; 3, 51–80%; and 4, >80%. The final staining score from 0–12 for ezrin and cortactin was the product of the staining intensity and positive area scores. A score of 0–7 indicated low expression and between 8–12 indicated high expression in ezrin. In cortactin, scores of 0–3, 4–7, and 8–12 were considered indicative of low, moderate, and high expression, respectively.