Paraffin-embedded specimens were sectioned to 5-µm thickness on to glass slides. In certain cases, tissue microarray slides were purchased from the commercial or academic sources as described in the results section. In all cases, the sections were subjected to antigen retrieval by heating the slides for 5 min in 5 mM sodium citrate and stained for the gene product using a specific antibody as previously described (14 (link)). Overnight incubations with primary antibodies at 4°C were followed by TRITC-conjugated (1:500; cat. no. 111-296-003) or FITC-conjugated (1:500; cat. no. 111-096-003; both from Jackson ImmunoResearch Laboratories, Inc.) secondary antibodies at room temperature for 1 h. The slides were then counterstained and mounted with 1.5 µg/ml DAPI (cat. no. H-1200, Vector Laboratories, Inc.) at room temperature. Controls were incubated either in the presence of no primary antibody, no secondary antibody, or primary antibody blocked with the antigen peptide.
Following this, the slides were observed under Nikon Optiphot 2 fluorescent microscope, and the images were captured by Retiga 1300R electronic camera, acquired on iMac computer using iVision image analysis program (BioVision, Inc.). The cells labelled with the fluorescent dye were counted as immunopositive cells whereas those stained with DAPI were counted for total cells in a field (magnification, ×400).
Following this, the slides were observed under Nikon Optiphot 2 fluorescent microscope, and the images were captured by Retiga 1300R electronic camera, acquired on iMac computer using iVision image analysis program (BioVision, Inc.). The cells labelled with the fluorescent dye were counted as immunopositive cells whereas those stained with DAPI were counted for total cells in a field (magnification, ×400).