IAV binds to secreted mucus in human trachea tissues. Frozen human trachea tissue sections were stained with Hematoxylin and Eosin (H&E), periodic acid Schiff (PAS, mucin staining in pink), Sambucus nigra agglutinin (SNA, binds to Siaα2-6Gal/GalNAc) or TKH2 antibody (binds to Sialyl Tn: Siaα2-6GalNAc on O-linked glycans). Dashed lines specify the location of secreted mucus, which is preserved in the frozen tissues, as seen in H&E and PAS staining. Both SNA and TKH2 bound to the secreted mucus (dark brown staining), indicating abundant potential ligands for IAV binding. TKH2 binding was confined to the lining of the epithelium and the glands, further confirming that the secreted mucus layer is adequately preserved in these tissues. Binding of IAV to the tissues was tested by incubating 600 HAU of virus 1.5 h at room temperature. All three strains bound to the secreted mucus layer (dashed line), and to ciliated cells (cells stained in dark brown). Virus was detected by anti-NP antibodies. Boxed area is enlarged below each image. Scale bar indicates 500 μm, scale bar of enlarged area indicates 50 μm.