P0720

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.