Immunohistochemistry on tissue specimens and tissue microarrays

All tumor samples were routinely fixed for 24 h in 4% buffered formaldehyde, dehydrated and then embedded in paraffin. Immunohistochemistry was performed on 5 µm sections that were mounted on silane-coated glass sides. Before starting the immunodetection, tissue sections were briefly exposed to microwave pre-treatment in a 0.01M citrate buffer (pH 6.2) for 7 min at 900 W and then for 10 min at 350W. The sections were then pretreated with 0.06% hydrogen peroxide solution (H₂O₂) for 4 min to block endogenous peroxidase activity, rinsed in phosphate-buffered saline (PBS) and successively exposed for 5 min to solutions containing avidin (0.1 mg/ml PBS) and biotin (0.1 mg/ml PBS) to avoid false-positive staining reaction from endogenous biotin. After a washing step with PBS, the sections were incubated for 15 min with a solution of 0.5% casein in PBS and sequentially exposed at room temperature to solutions of i) specific primary antibody (1 h): Galectin-1 (polyclonal rabbit anti-human galectin-1: 1:100) (29,37), Galectin-3 (polyclonal rabbit anti-human galectin-3: 1:200) (29,37), CK19 (monoclonal mouse anti-human cytokeratin 19: 1:50; M0772; Dako, Glostrup, Denmark); ii) corresponding biotinylated secondary antibody (30 min): polyclonal goat anti-rabbit/mouse IgG (1:50; BA-1000/BA-9200, Vector Laboratories, Burlingame, CA, USA); and iii) avidin-biotin-peroxidase complex (ABC Kit Vector Laboratories). The specificity of galectin-1 and -3 antibodies was validated by western blotting in different thyroid cancer cell lines (B-CPAP, FTC133C and 8505C cell lines derived from papillary, follicular and anaplastic thyroid carcinoma respectively) reporting immunoreactive band for galectin-1 at 14 kDa and for galectin-3 at 26 kDa (data not shown). The slides were thoroughly washed with PBS between incubation steps. Immunocomplexes were finally visualized by exposure to the chromogen diaminobenzidine (DAB, BioGenex, Fremont, CA, USA) in the presence of H₂O₂. After rinsing, the sections were counterstained with luxol fast blue and mounted with a synthetic medium. To exclude antigen-independent staining, the incubation step with primary antibodies was omitted from the protocol as negative controls. In all cases, these controls were negative (data not shown). Individual tissue specimens were given a score (0–6) by adding the percent of immunopositive cells (range 0–3: 0=0%, 1=1–33%, 2=34–66%, and 3=67–100%) to the intensity score (range 0–3, 0=no, 1=low, 2=moderate, and 3=high).