Retinal Cross-Sections Preparation and Analysis

Agarose-embedded retinal cross-sections were prepared as described^{55 (link)}, collected in 24-well plates, and incubated for 2 h with Alexa Fluor 594 conjugate of wheat germ agglutinin (Invitrogen) in PBS containing 0.1% Triton X-100. Sections were washed three times in PBS, mounted with Fluoromount G (Electron Microscopy Sciences) under glass coverslips, and visualized using a Nikon Eclipse 90i confocal microscope. GFP fluorescence was excited at 488 nm. Plastic-embedded retinal cross-sections (1 μm) were prepared as described in refs. ^{9 (link),56 (link)} and stained with toluidine blue for light microscopy. Nuclear count in 100 μm segments of the outer nuclear layer was performed in sections cut through the optic nerve at 500-μm steps from the optic nerve head. Tangential sectioning of flat-mounted frozen retinas was performed as described in refs. ^{22 (link),56 (link)}.