Immunohistochemistry. The eyes and

Immunohistochemistry. The eyes and adnexa (four eyes per group) were surgically excised, fixed in 4% buffered paraformaldehyde solution overnight at 4∘C, and paraffinembedded. Then, the oxidative stress-induced lipid peroxidation marker was evaluated by immunohistochemically detecting4-hydroxy-2-nonenal(4-HNE)intheconjunctiva. Six-micrometer sections were cut from the paraffin wax blocks, mounted on precoated glass slides, deparaffinized, and rehydrated. Then, 0.3% hydrogen peroxide (H2O2) and 1% serum both in PBS were sequentially applied to conjunctival sections, which were then incubated with mouse anti-4-HNE monoclonal antibody (JaICA, Shizuoka, Japan) at a concentration of 25g/mL for 1h at room temperature. After washing the sections, they were incubated with the rabbitanti-mouseIgGsecondaryantibodies.Then,thetissue sectionswereincubatedwithavidin-peroxidase,followedby the 3,3-diaminobenzidine peroxidase substrate, and then counterstained with Mayer’s hematoxylin. The number of positivelystainedcellsfor4-HNEper100mwascounted.