Maackia amurensis agglutinin 2 maa 2

Influenza virus receptors on cultured cells were characterized using FITC-labelled Sambucus nigra agglutinin (SNA) (Vector Labs) for SAα 2,6 Gal, and biotinylated Maackia amurensis agglutinin II (MAA II) (Vector Labs) for SAα 2,3 Gal in a previously described lectin-cytochemical method [20] .

Cells were grown on 8-well Lab-Tek II chamber slides (Nunc). Lectin labelling was performed as previously described [15 (link)]. Cells were fixed with 4% paraformaldehyde for 10 min at room temperature, after which endogenous biotin activity was blocked using a streptavidin/biotin blocking kit (Vector Laboratories). Cells were incubated with fluorescein isothiocyanate (FITC)-labelled Sambucus nigra agglutinin (SNA), specific for human influenza receptor type SAα2,6-Gal, and biotinylated Maackia amurensis agglutinin II (MAA II) (Vector Laboratories) specific for avian influenza receptor type SAα2,3-Gal overnight at 4 °C, at 10 μg/ml each. After overnight incubation, cells were washed twice with Tris-buffered saline (TBS) and subsequently incubated with streptavidin-Alexa Fluor 594 conjugate (Invitrogen) at room temperature for 2 h. Finally, cells were washed three times with TBS and mounted with ProLong Gold antifade reagent with 4′,6′ diamidone-2-phenylindole (DAPI) (Invitrogen). Neuraminidase derived from Clostridum perfringens (11,585,886,001; Roche) was used at 0.05 U/ml in culture medium for 4 h at 37 °C for the collective removal of SA receptors [16 (link)].