Ocular histology

Eyes were harvested at appropriate time points and fixed in half strength Karnovsky fixative (4% paraformaldehyde + 2.5% glutaraldehyde) in 0.1M PO₄ buffer pH 7.4 for 48 hours, dehydrated in graded ethanol and embedded in Technovit 7100 Glycol Methacrylate (Electron Microscopy Sciences, Hatfield, PA). Embedded tissues were serially sectioned (2 μm) by passing through the optic nerve head and stained with Hematoxylin and Eosin (H&E). For each retina a section cut along the naso-temporal equator containing the optic nerve head was acquired by an upright Axiophot microscope (Zeiss, Germany) equipped with a Plan Neofluar 20X objective (NA 0.5). A blinded investigator quantified the thickness of the outer and inner nuclear layers and of the inner plexiform layer at ten locations equally spaced along the naso-temporal axis, and values for a given retina were expressed either as average number of nuclei (for nuclear layers) or average microns (for the inner plexiform layer).