At the end of any treatment, corneas were dissected. Epithelium was removed after EDTA (Sigma-Aldrich Corp., St. Louis, MO, USA) treatment (30 minutes, 37°C). Subsequently, the stroma was fixed and immunostained as previously described
34 for the following markers: rat anti-CD31 (blood vessel marker; BioLegend, San Diego, CA, USA), goat anti-LYVE1 (lymphatic marker; AbCam, Cambridge, UK), and rabbit anti-CD45 (leukocyte marker; R&D Systems, Minneapolis, MN, USA). Corneas were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Inc., Burlingame, CA, USA) flat-mounted, and photographed by epifluorescence microscope (Leica CTR5500; Leica Microsystems, Wetzlar, Germany). A set of six adjacent, overlapping images were acquired and remapped into a montage, obtaining a two-dimensional (2D) reconstruction of the whole cornea. Digital pictures were analyzed using ImageJ software (
http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA), and the neo-vascularized area of the cornea was encircled by drawing a freehand region: the CNV index was calculated as the difference between the total corneal area and the avascular area, normalized for the total corneal area.
Immune cell infiltration was quantified by counting the CD45+-positive cells per field; six peripheral fields (40×, 5-μm z-stack) per cornea were taken with a confocal microscope (Leica TCS SP5; Leica Microsystems).
To evaluate the innervation rate in different mouse strains, freshly collected corneas were processed and immunostained for the nerve marker TUJ1 (rabbit anti–β-3-tubulin polyclonal antibody; Chemicon, Burlington, MA, USA), as previously described.
35 Six peripheral fields of the peripheral subbasal nerve plexus (40×, 5-μm
z-stack) per cornea were taken with confocal microscope (TCS SP5; Leica Microsystems), and the total nerve length was calculated with neuronJ.
To evaluate corneal thickness, murine eyes were frozen in OCT solution (Killik; Bio-Optica, Milano, Italy) and stored at −80°C until cross-sectioning (8 μm) and staining with hematoxylin-eosin; thickness was measured with ImageJ software (National Institutes of Health).