The polynuclear eosinophil leukocytes were stained with Direct Red and counted in the venous plexus region of the sclera. Immediately after excision, the eyes were embedded in a protective tissue freezing medium (Tissue Tek OCT compound; Sakura Finetek, Inc., Torrance, CA), frozen in liquid nitrogen, and stored at −80°C. Then, 6 μm thick slices were prepared with a cryostat and fixed in cold acetone for 10 minutes. After being dried, the slices were rehydrated by successive baths in toluene (5, 3, and 2 minutes), then in a 100% ethanol solution (3 and 2 minutes), a 95% ethanol solution (3 and 2 minutes), and a 50% ethanol solution (2 minutes). The sections then were bathed for 20 minutes in a staining solution of 0.03% Sirius red in 50% ethanol (Direct Red 75 dye content 30%; Sigma-Aldrich), rinsed with running water for 5 minutes, and mounted in an aqueous medium (glycerol/PBS, 50/50 vol/vol).
The eosinophils, bright pink stained on an illuminated background, were counted in the venous plexus region of the sclera under a Nikon Eclipse 90 I microscope equipped with a Nikon DXM1200F digital camera (both from Nikon Instruments Inc., Melville, NY). The area of the zone to be counted was determined with Nikon Lucia image analysis software (release 4.8; Nikon Instruments Inc.) and counts were expressed as the number of eosinophils per mm2.
The results obtained for the four experimental groups were compared using a one-way ANOVA, followed by a Bonferroni multiple comparison test with statistical significance set at P < 0.05.