Fig 1: Confocal images of sensory nerves immunostained with anti–β tubulin III antibody in mouse cornea. (A) Sensory nerve trunks enter from the limbus into the stroma of the cornea where they ramify, giving rise to a dense subepithelial plexus (asterisks). (B) Detail of a stromal nerve trunk branching. (C,D) From the stroma, nerve fibers penetrate through the basal lamina (arrowheads) and form the subbasal plexus. Subbasal nerve fibers run parallel for a long distance within the epithelium basal cell layer. (E–I) Subbasal nerves give rise to terminal branches that ascend along their trajectory through the epithelial cells. According to the number of branches, three morphological types of corneal nerve terminals are identified: simple (G), ramified (H), and complex (I) nerve terminals. Scale bars: (A) 250 µm; (B) 25 µm; (C–F) 50 µm; (G–I) 10 µm. Methods: C57BL/6J eyes were fixed for 2 h at RT in methanol and DMSO (4:1), incubated 5 min at −20 °C in methanol, rehydrated, and washed in PBS. (A–F) Corneas were dissected, incubated 2 h at RT in blocking solution (5% goat normal serum, 1% BSA and 0.1%Triton X–100 in PBS) and 48 h at 4 °C with anti–β tubulin III antibody (1:500 in blocking solution; #801201, BioLegend, CA, San Diego, USA), rinsed and then incubated for 2 h at RT with AF555 (1:500 in PBS; #A32727, ThermoFisher Scientific, OR, Waltham, USA), washed in PBS and mounted with Fluoromount-G (ThermoFisher Scientific). (G–I) Eyes were cryoprotected (30% sucrose in PBS overnight at 4 °C), embedded in OCT, frozen in liquid nitrogen, cut on a cryostat in serial 15 mm thick sections, and mounted in slides. Tissue sections were rinsed in 0.03% Triton X–100 in PBS and then for 30 min in blocking solution followed by overnight incubation at 4 °C with anti–β tubulin III antibody in blocking solution, washing with PBS and incubation with AF555 in PBS for 2 h at RT. Afterward, slides were rinsed with PBS, incubated with Hoechst 33342 (10 μm/mL; #H1399, ThermoFisher Scientific), and coverslipped with Fluoromount–G. Images were collected using a laser scanning confocal microscope Zeiss LSM 880 (Oberkochen, DEU).