Three-month-old 129Sv, Gαi3 −/−, B6/NCrl, and Oa1 −/− mice were anesthetized by intraperitoneal injection of 120 mg/kg sodium pentobarbital and were perfused intracardially with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4. Eyes were enucleated, rinsed in 0.1 M phosphate buffer, postfixed with 1% buffered osmium tetroxide, dehydrated in graded ethanol, and embedded in araldite 502. Sections for light microscopy (1 μm) were photographed with a digital camera (CoolSNAP; Roper Scientific, Duluth, GA) attached to a microscope (210; Zeiss, Thornwood, NY). Images at 100× magnification were merged with the use of software (Image-Pro Plus; Media Cybernetics, Bethesda, MD). For ultrastructural analysis, 60- to 70-nm stained sections (5% uranyl acetate and 0.4% lead citrate) were observed with an electron microscope (EM 910; Zeiss), and RPE fields were photographed with a digital camera (Keenview). The number of melanosomes and their areas in the RPE of 129Sv, Gαi3 −/−, B6/NCrl, and Oa1 −/− mice were analyzed on 30 random micrographs per line at 16,000× magnification using software (analySIS 2004; Soft Imaging System, Muenster, Germany). Starting at the top left of each section, fields were scanned serially to the right until the whole grid surface was covered. To determine the size and number of melanosomes, first each micrograph was loaded into the software platform (Soft Imaging System); then individual melanosomes were selected with the magic wand tool to measure the area, and the program produced two spreadsheets per micrograph containing the melanosomal count, the individual areas, and their mean size and SD. In total, 1540, 1115, 1550, and 530 melanosomes were analyzed for 129Sv, Gαi3 −/−, B6/NCrl, and Oa1 −/−, respectively. Images were automatically contrast enhanced with Adobe Photoshop (CS2 9.0.2; Adobe Systems Inc., San Jose, CA). Melanosome densities in the Oa1 −/− and Gαi3 −/− mice, and their respective control mice, were compared using Student’s t-tests (P < 0.05 was considered significant), and the distribution of melanosomes by size as a function of their frequency in control and knockout mice was displayed in histograms.