Flagellin Treatment of Mice

Wild-type mice were treated topically with 10 ng flagellin (InvivoGen, San Diego, CA) bilaterally (5 µl/eye) for 7 days. Corneal barrier integrity was monitored for 4 weeks by fluorescein staining as described previously (12). Briefly, 1% sodium fluorescein (Sigma-Aldrich, St. Louis, MO, USA) was applied to each eye. Three minutes later, eyes were flushed with PBS to remove excess fluorescein, and corneal staining was evaluated using the slit-lamp microscope (Haag-Streit, USA) with cobalt blue light. Punctate staining was scored according to the standardized National Eye Institute grading system of 0–3 for each of the five areas of the cornea (13).