Figure 2. Representative histology stains and fluorescent imaging of pseudo-ALI cultures.

A. Histology stains of a representative nasopharyngeal pseudo-ALI culture. Shown is an uninfected pseudo-ALI culture grown from a donor collected at the time of skull base surgery. H&E, hematoxylin and eosin stain shows a pseudostratified epithelium with columnar ciliated cells. Ki67, nuclear protein Ki67 is a proliferation marker that marks cycling cells located in the basal layer. AB/PAS, Alcian blue/periodic acid-Schiff stains acidic and neutral mucins, marking different types of mucosecretory cells. B. Fluorescent Z-stack images of EBV-infected cells in pseudo-ALI culture showing GFP-positivity (green), counterstained with DAPI (blue). The pseudo-ALI culture was infected with an epithelial-tropic recombinant EBV strain (M81, Tsai et al., 2013 ) that co-expresses GFP, fixed in 4% paraformaldehyde at 4 days post-infection (p.i.), and imaged by confocal microscopy. 3-D renderings were assembled in Nikon NIS Elements. EGF treatment (10 ng/mL) enhances cell-free virus infection ( Wang et al., 2015 ). If live cell imaging is preferred, the images would have to be captured directly in the transwell (without mounting of the excised membrane), and a suitable objective with an extended focal length would have to be determined by the end user. Note that the B-cell co-culture infection method would be clouded by GFP-positivity from input B-cells, and is, therefore, not recommended as a method for assessing infection.