Barrier function assays
The TJ permeability assay was performed according to previous study by
using surface biotinylation25. Briefly, ALI-cultured
hNECs were incubated with 1 mg/ml EZ-Link Sulfo-NHS-LC-biotin (557 Da;
Thermo Fisher Scientific, Inc., Waltham, MA) in DPBS for 45 min. After
washing, the samples were fixed overnight with 4% PFA at 4°C, embedded
in paraffin, and sectioned at a thickness of 4 µm. After antigen
retrieval, sections were washed and blocked with goat serum, and then
incubated for 30 min with streptavidin (Alexa Fluor™ 488 conjugate,
Thermo Fisher Scientific).