Immunofluorescence Staining of Whole-Mount Preparations

The normal corneas of healthy mice and the left corneas of neurotrophic keratopathy model mice killed 14 days after axotomy in the right eye were excised, washed in PBS(–), and fixed in acetone for 15 minutes at room temperature. The corneas were then incubated for 90 minutes at room temperature in PBS(–) containing 3% BSA in order to block nonspecific staining before exposure (overnight at 4°C) to primary antibodies in PBS(–) containing 3% BSA. The antibodies included NL637-conjugated monoclonal anti-βIII-tubulin (1:100 dilution; R&D Systems, Minneapolis, MN, USA), the polyclonal SP antibody N-18 (1:50 dilution, sc-9758; Santa Cruz Biotechnology, Dallas, TX, USA), and the polyclonal NK-1R antibody (1:50 dilution, NB300-101; Novus Biologicals, Centennial, CO, USA). The tissue was washed three times for 5 minutes with PBS(–), incubated for 1 hour at 4°C with Alexa Fluor 488 conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA) at a 1:2000 dilution in PBS containing 3% BSA, and then washed again. Corneal whole-mounts were prepared with VECTASHIELD mounting medium containing propidium iodide (PI; Vector Laboratories, Burlingame, CA, USA). Central and peripheral nerves of the whole-thickness cornea were imaged in z-axis steps of 2 mm with a laser confocal microscope (LSM Pascal; Carl Zeiss Meditec, Jena, Germany).