Animals were euthanized in the morning, between 10:00 AM and 12:00 noon, by being administered a lethal dose of pentobarbital. After marking the dorsal margin of the limbus with a stitch, eyes were enucleated and fixed in 4% (w/v) paraformaldehyde for 1 hour at room temperature (RT). After being washed in PBS, eyes were cryoprotected in 15%, 20%, and 30% sucrose. The cornea, lens, and vitreous body were removed, and the eyecups were processed for vertical sections or whole mounts. For cryostat sections, eyecups were frozen in optimal cutting temperature compound with liquid N2. Sections (14 μm thick) were then obtained using a commercial cryostat (Leica CM 1900; Leica Microsystems, Wetzlar, Germany), mounted on slides (Superfrost Plus; Gerhard Menzel Glasbearbeitungswerk GmbH & Co. KG, Braunschweig, Germany), and air-dried. Sections were incubated in normal donkey serum diluted 1:100 in PBS containing 0.5% Triton X-100 for 1 hour, and then subjected to single immunostaining overnight at RT, using rabbit antimelanopsin antibody 1:2000 in PBS containing 0.5% Triton X-100. After several washes in PBS, Alexa Fluor 488 (green)–conjugated donkey anti-rabbit IgG secondary antibody was applied at a 1:100 dilution in PBS containing 0.5% Triton X-100 for 1 hour. The sections were finally washed in PBS, mounted in antifadent mounting medium (Citifluor; Citifluor Ltd., London, UK), and coverslipped for laser-scanning confocal microscopy viewing (Leica TCS SP2 system; Leica Microsystems). Immunohistochemical controls were performed by omission of either the primary or secondary antibodies. Final images from control and experimental subjects were processed in parallel, using photo-editing software (Adobe Photoshop 10.0; Adobe Systems, Inc., San Jose, CA).