Detailed procedures for the immunohistochemical assays and scoring system are described elsewhere [36 (link), 37 (link)]. Briefly, each tissue slide was reacted with monoclonal antibodies against Slug (Cell Signaling Technology); fascin (Santa Cruz Biotechnology); or CLDN-1, -2, or -3 (Novus Biologicals) in a humidified chamber for 60 min at 37°C. Afterwards, the sections were washed with PBS and treated with an appropriate biotin-conjugated secondary antibody for 30 min. The signal for each tissue section was detected using the avidin-biotin complex system and diaminobenzidine kit (Vector Laboratories, Burlingame, CA, USA). Immunohistochemical staining was quantified according to the coverage area and intensity for Slug as described [38 (link)]. The scoring system for the CLDNs and fascin is described elsewhere [39 (link)], with the following modifications: –, no staining or membrane staining in < 10% of tumor cells; +, faint, weakly perceptible positive staining of the membrane in < 10% of tumor cells; ++, weak-to-moderate complete membrane staining observed in ≥ 10% of tumor cells; and +++, strong, complete membrane staining in ≥ 10% of tumor cells.
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