To quantify and visualize RGCs, the whole-mount retinas were incubated for 6 days at 4 °C with primary antibody against the mouse RNA-binding protein with multiple splicing (RBPMS; 1:1000 dilution, guinea pig anti-RBPMS, PhosphoSolutions, Aurora, CO, USA), which is uniquely expressed in RGCs [19 (link)]. This was followed by incubation with 1:400 Alexa Fluor® 488 conjugated rabbit anti-GFP (Molecular Probes, Eugene, OR, USA) and Cy3 conjugated donkey anti-guinea pig secondary antibody (Jackson Immuno Research Laboratories Inc., West Grove, PA, USA) overnight at 4 °C. After that, the retina was rinsed in PBS for 10 min, then incubated in the nuclear counterstain TO-PRO-3 iodide (Thermo Fisher Scientific, Waltham, MA) for 15 min. Retinas were flattened with RGCs facing up, mounted with anti-fade fluorescent mounting medium (Sigma-Aldrich, St. Louis, MO), and coverslipped. Images were taken using a × 20 objective with a confocal microscope (Nikon C1, Nikon Canada Inc., Toronto, ON). Three images with an area of 330.32 × 330.32 μm from each retina were used for RGC quantification: near the optic disk, near the periphery and at an intermediate distance. Image J was used to perform RGC counts.
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