At various points post-inoculation (pi), HAE were fixed in cold methanol-acetone (50/50) and stored at 4uC. Cultures were then permeabilized with 2.5% triton-X 100/PBS++ (containing 1 mM CaCl 2 and 1 mM MgCl 2 ) and blocked with 3% bovine serum albumin (BSA) in PBS++ before being probed with mouse antiinfluenza virus nucleoprotein (NP; Chemicon, Inc.; 1:100) and immunoreactivity detected with fluorescein isothiocyanate (FITC)conjugated anti-mouse IgG secondary antibody (Jackson Immu-noResearch Laboratories, Inc., 1:500). Fluorescent images were obtained using a Leica DMIRB inverted fluorescence microscope equipped with cooled-color charge-coupled-device digital camera (MicroPublisher; Q-Imaging, Burnaby, BC, Canada). The percentage of the epithelium positive for viral antigen as an index of percentage of infected cells was quantified over 5 images per culture by black and white pixilation of each image and computer calculation of percent black pixels after inverting the image. This technique determines percentage of black pixels in a defined area and does not account for differences in fluorescent intensity.