Alexa fluor 594 conjugate of wheat germ agglutinin

Agarose-embedded retinal cross sections were prepared as previously described (Lobanova et al., 2010 (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.
Plastic-embedded retinal cross sections (1 μm thick) were prepared as previously described (Sokolov et al., 2004 (link)) and stained with toluidine blue for light microscopy. Tiled images of whole retina cross sections were obtained using the Olympus IX-81 Inverted Fluorescence Microscope, and aligned and stitched using the Olympus cellSens Dimension software. The number of photoreceptor nuclei in representative segments of outer nuclear layer (ONL) was quantified as a quantitative measure of surviving photoreceptors. The number of nuclei in a 400 μm segment of the ONL, located at 1 mm from each side of the optic nerve, was counted by hand.

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)}.