MIF protein expression levels in normal and vascularized corneas were evaluated by Western blot analysis and immunohistochemistry. For Western blot analysis, the eyes were enucleated, and the corneal samples were placed into 150 mL of lysis buffer (20 mM imidazole HCl, 10 mM KCl, 1 mM MgCl2, 10 mM EGTA, 1% Triton, 10 mM NaF, 1 mM sodium molybdate, and 1 mM EDTA [pH 6.8]) supplemented with a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) and sonicated. The lysate was centrifuged at 14,000 rpm for 15 minutes at 4°C. The samples were boiled for 5 minutes and separated by SDS-polyacrylamide gel electrophoresis under denaturing conditions, and electroblotted to a polyvinylidine difluoride (PVDF) membrane (Bio-Rad, Hercules, CA). The membranes were incubated in blocking buffer, followed by the rabbit anti-MIF polyclonal antibody (Novus Biologicals, Littleton, CO), and then washed and incubated with a horseradish peroxidase-labeled anti-rabbit antibody (GE Healthcare, Piscataway, NJ). The blot was visualized with an ECL Plus kit (GE Healthcare) according to the manufacturer’s instructions. NIH Image was used for the quantification of band density.
For immunohistochemistry, eyes were enucleated and embedded in OCT compound, snap frozen in liquid nitrogen, and cut into 7-μm-thick sections. The sections were applied with rabbit anti-MIF polyclonal antibody as the primary antibody at 2 μg/mL at room temperature for 1 hour. For the negative control, nonimmunized serum (Vector Laboratories, Burlingame, CA) was used in place of the primary antibody. Immunoreactivity was detected with a kit (Histofine SAB-PO; Nichirei, Tokyo, Japan), according to the manufacturer’s protocol. Briefly, the samples were incubated with biotinylated anti-rabbit goat serum for 15 minutes at room temperature and then rinsed with PBS, after which they were incubated with a streptavidin-biotin-peroxidase complex for 10 minutes at room temperature. The final reaction product was visualized with 3,3′-diaminobenzidine tetrahydrochloride (DAB). Counterstaining was performed with hematoxylin.