On days 3 and 5 after RD creation, the eyes were enucleated and embedded in OCT compound (Tissue Tek; Sakura Finetec, Torrance, CA). Serial sections of the eyes in the sagittal plane through the optic nerve head were cut at 10 μm thickness on a cryostat (CM1850; Leica, Heidelberg, Nussloch, Germany) at −20°C and prepared for staining. After fixation in acetone, endogenous peroxidase activity was blocked by 0.3% hydrogen peroxide (Sigma-Aldrich, St. Louis, MO) for 15 minutes and subsequently with 5% skim milk for 1 hour, to block nonspecific binding. Subsequently, sections were incubated with anti-4-hydroxynoneal (HNE) Ab (Alpha Diagnostics, San Antonio, TX) at 4°C overnight. Thereafter, sections were incubated for 30 minutes at room temperature with HRP-conjugated secondary antibody against rabbit IgG (Dako, Carpinteria, CA). For signal detection, the sections were incubated with AEC substrate-chromogen solution for 5 minutes according to the manufacturer's protocol (Envision System-HRP; Dako) and then washed with distilled water. Finally, the sections were counterstained with Mayer's hematoxylin (Sigma-Aldrich). Light microscopy was used to obtain images of the retina at a final magnification of ×200.