Biotinylated sna

The lectin reactivity of the TF-specific IgG was measured by ELISA in a similar way, except that the binding of neuraminic acid- (sialic acid-) specific Sambucus nigra agglutinin (SNA) and mannose-specific concanavalin A (ConA) to the absorbed serum anti-TF Abs (all isotypes) or anti-TF IgG from tIgG samples was measured as described elsewhere [19 (link), 21 (link)]. Biotinylated SNA (Vector Laboratories Inc., USA) in 10 mmol/L HEPES, 0.15 mol/L NaCl, and 0.1 mmol/L CaCl₂, pH 7.5, and biotinylated ConA (Sigma, USA) in the ConA binding buffer (0.05 mol/L Tris-HCl buffer, pH 7.2, containing 0.2 mol/L NaCl and 3 mmol/L CaCl₂, MgCl₂, and MnCl₂) were both applied at a concentration of 5 μg/mL each, for 1.5 h at 25°C. The bound lectins were detected with a streptavidin-alkaline phosphatase conjugate (Dako, Denmark) and p-nitrophenylphosphate (Sigma, USA). The optical density value of control wells (no sample) was subtracted from that of Ab-coated wells to determine the lectin binding. Each sample was analyzed in duplicate.

Tissue sections were baked at 60 °C for 45 min followed by deparaffinization with xylene and rehydration with graded alcohols. Antigen retrieval was done by heating slides in a pressure cooker for 3 min in citrated buffer (pH 6.0, 10 mM trisodium citrate). After cooling down at room temperature, tissue sections were incubated with 3% hydrogen peroxide for 10 min. For neuraminidase treatment, slides were incubated at 37 °C for 3 h in 5 mM CaCl and 50 mM ammonium acetate containing 2000 U/mL of neuraminidase (New England Biolabs P0720) and then blocked with 5% normal goat serum or BSA in Tris-buffered saline with 0.1% Tween-20 (TBST) for 1 h at room temperature. Slides were incubated with primary antibodies (5 μg/ml) or biotinylated SNA (20 μg/ml) (Vector Labs) ON at 4 °C. Next, slides were incubated with HRP-conjugated anti-rabbit, anti-mouse or streptavidin secondary antibodies (KPL Inc., Gaithersburg, MA) at room temperature for 1 h. Signals were visualized by incubating sections with Aminoethylcarbazole (AEC) substrate solution (Invitrogen), and cell nuclei were counterstained with hematoxylin (Invitrogen). Slides were imaged on ImageXpress Pico Automated Cell imaging system (Molecular Devices). Human tissue sections mounted on glass slides in 5-μ sections were obtained from US Biomax, Rockville, MD.

Lectin-histochemistry staining to detect MAL-SG and SNA-SG in CCA tissue was processed as previously described [3 (link)]. In brief, CCA tissue sections were de-paraffinized, re-hydrated, and incubated with 40 µg/mL of biotinylated-MAL-II and 1 µg/mL biotinylated-SNA (Vector Laboratories, Burlingame, CA, USA), respectively. Negative control slides were incubated with phosphate buffer saline (PBS) instead of biotinylated-lectin. Expression of MAL-SG and SNA-SG in CCA tissues was semi-quantified as a MAL-SG score and a SNA-SG score, according to their staining intensity (0, negatively stained; 1+, weakly stained; 2+, moderately stained; and 3+, strongly stained) and frequency of each intensity (% of total area) based on the H-Score system [29 (link)].