Peroxidase kit

Paraformaldehyde fixed and paraffin embedded tissues were cut into 5 μm sections, subjected to sodium citrate antigen retrieval, and stained using the M.O.M. Peroxidase kit (Vector Labs, Burlingame, CA) and 3,3′-Diaminobenzidine (DAB). The following antibodies were used: DEK (1:60, BD Biosciences), BrdU (1:100, Molecular Probes, Invitrogen, Grand Island, NY), Wnt10b [H70] (1:50, Santa Cruz), MMP-2 and MMP-9 (1:100 and 1:200, respectively, Santa Cruz), cytokeratin-5 (1:200, Abcam), or activated β-catenin [8E7] (1:100, Millipore). Samples were counterstained with 0.1% Nuclear Fast Red (Poly Scientific, Bay Shore, NY) and preserved with Permount (Fisher Scientific, Pittsburgh, PA). Tissue microarrays were purchased from Imgenex (CBA2 and CBB2; Imgenex, San Diego, CA) for correlations of Ron, DEK, and β-catenin expression in human tissues and scored as previously described.^{6 (link)} For R7 xenograft tumors, staining intensity was quantified by the Threshold tool on Image J. The threshold for each section was set and normalized to each section’s respective total area of tissue as seen at 200x total magnification. Scores were calculated by multiplying the intensity score by the percentage of positive staining cells. The total number of cells counted per section ranged from 122 to 357. For activated β-catenin staining, cells were considered positive if the nucleus showed DAB staining.