Detection of a2,3 and a2,6 linked sialic acids HAE maintained at either 32uC or 37uC for 72 hrs prior to sialic acid detection were washed, blocked with 3% BSA/PBS++ and probed with biotinylated SNA or MAA lectins to detect a2,6 and a2,3 SA, respectively (Vector Laboratories, Inc.; EY-Laboratories, Inc.; 1:100). HAE were then fixed in 4% PFA and incubated with streptavidin-alexafluor 488 (Molecular Probes, Inc.; 1:500) applied to the apical surface to detect lectin binding. Immunohistochemistry HAE fixed in methanol:acetone, were probed en face with antibody against viral NP (Chemicon, Inc.; 1:100) and FITCconjugated goat anti-mouse IgG1 and IgG2a (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA; 1:500), then embedded in paraffin. Histological sections (5 mm) were prepared and reprobed for viral antigen using standard immunofluorescence protocols. Briefly, sections were bathed in 2.5% triton-X 100/ PBS++ for 30 min, blocked in 3% BSA/PBS++ and incubated with antibodies in 1% BSA/PBS++. Primary antibodies were antiviral NP (Chemicon, Inc., as above) and anti-alpha acetylated tubulin (Zymed Laboratories, Inc.; 1:2000), a marker for ciliated cells. Secondary antibodies were FITC-goat anti-mouse IgG2a and Rhodamine red-conjugated goat-anti-mouse IgG2b (Jackson ImmunoResearch Laboratories, Inc.; 1:500). Sections were prepared with FluorSave mounting media (EMD Chemicals, Inc.) and images captured using a Leica DMIRB inverted fluorescence microscope equipped with a cooled color chargecoupled-device digital camera (MicroPublisher; Q-Imaging, Burnaby, British Columbia, Canada).