Ten mouse corneas were used from each genotype to measure expression of occludin in +/+, +/−, −/−, and −/− mice on the special diet. Tissue was collected and stored in a protease inhibitor solution at −80°C. Corneas were freeze-dried with liquid nitrogen and crushed. Lysis buffer D at 95°C (0.3% SDS, 10 mM Tris/HCL, 10 μM sodium orthovanadate, 100 μM sodium fluoride, and protease inhibitor) was added to the tissue, which was collected for further processing. The mixture was sonicated five times in ice water followed by incubation at 95°C for 10 minutes, and centrifuged at 18187g (4°C) for 10 minutes followed by supernatant recollection. Sample protein concentration was measured using the bicinchoninic acid protein assay reagent (Pierce, Rockford, IL, USA). Equal amounts of protein (20 μg) were loaded onto an 8% gel and separated by SDS-PAGE. Mouse anti-occludin, (Zymed; Invitrogen, Carlsbad, CA, USA) and a horseradish peroxidase conjugated goat anti-mouse secondary antibody was used to enhance detection. For loading controls, membranes were stripped and re-probed with B-actin antibody (CP01; Calbiochem, Billerica, MA, USA). Western blots were digitally photographed and blot density was determined by Fiji (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA).