Vectastin elite abc reagent

Protein expression was evaluated as previously described (22 (link)–24 (link)). Exponentially growing cell lines were plated on a glass chamber slide (Nunc Inc., Naperville, IL, USA) at a density of 1×10⁴ cells/ml of medium and allowed to grow for 2–3 days until they reached 70% confluence (21 (link)). The cells were fixed with buffered paraformaldehyde at room temperature, incubated with 1% H₂O₂ in methanol to block endogenous peroxidase and washed twice with buffer solution. Cell cultures were subsequently covered with normal horse serum for 30 min at RT and incubated with anti-rabbit monoclonal antibody (vimentin: C-20, sc 7557 and Notch 4: C-19, sc 8644) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a 1:500 dilution at 4ºC overnight, and then incubated for 45 min with diluted biotinylated secondary antibody solution (Vector Laboratories, Burlingame, CA, USA) and Vectastin Elite ABC Reagent (Vector Laboratories). The experiments were repeated three times in cells with identical passages in vitro. The number of immune-reactive cells (50 cells/field) was counted in several randomly selected microscopy fields (×400) per sample using an optical microscope (C×31; Olympus Corporation, Tokyo, Japan). Ten fields were counted for each cell line.