Quantitative Analysis of Corneal Opacity and Vascularization

Corneal opacity was quantitatively assessed as previously described [29 (link)] with tissue assayed for absorbance at 500 nm using a FLUOstar Omega plate reader (BMG Labtech, Offenburg, Germany) as previously described [59 (link)]. Following the measurement of cornea opacity, the corneas were fixed in a 4% solution of paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 30 min and washed in PBS containing 1% Triton X-100 (Sigma-Aldrich) [29 (link)]. The tissue was then blocked overnight in 10% donkey serum (Abcam, Boston, MA, USA) and labeled for blood and lymphatic vessels as previously described [42 (link)]. Image acquisition was obtained using an Olympus FV1200 scanning confocal microscope in sequential scanning channel mode (Center Valley, PA, USA). The total area positive for blood and lymphatic vessels per field of view (4 quadrants/cornea) was quantified using Metamorph software (Molecular Devices Inc., San Jose, CA, USA).