Fixation and Sectioning of Airway Epithelium

At 4 days postinfection (DPI), 200 μl of 1x PBS was added to the apical site of the Transwell for washing before PFA fixation. In the basal side of the Transwell inserts was 200 μl of 1x PBS. For PFA fixation, 200 μl of 4% PFA (Polysciences) was added to the upper chamber of the Transwells and incubated for 30 min, and the Transwells were further maintained overnight in 4% PFA prior to removal from high biocontainment. The PFA fixation protocol was approved as an inactivation method for coronaviruses by the RML Institutional Biosafety Committee. The PFA-fixed airway epithelium was paraffin-embedded and sectioned at a thickness of 5 μm for slide preparation as previously described [43 (link)].