All procedures, where possible, were conducted in low-level lighting so as not to bleach the fluorescent signal of the tdTomato reporter protein. After euthanasia, whole globes were enucleated and emersion fixed in 4% paraformaldehyde in PBS (50 mM phosphate buffer, pH 7.4, 150 mM NaCl) at 22°C between 30 and 60 minutes. Globes were then dissected to remove the anterior chamber and lens, and the resulting eyecup was incubated in 0.4% paraformaldehyde in PBS, overnight at 4°C. Eyecups were then equilibrated in 30% sucrose in PBS and embedded in optimum cutting temperature compound (Tissue-Tek; Sakura Finetek USA, Inc., Torrance, CA, USA). Sections were cut at 10 μm and affixed to Superfrost Plus microscope slides (ThermoFisher Scientific, Waltham, MA, USA). Sections were chosen for analysis that contained substantial portions of the optic nerve. Sections were blocked in PBS containing 2% BSA and 0.1% Triton X-100 overnight at 4°C in humidified chambers. Primary antibodies were then applied in the same buffer for 48 hours at 4°C. The primary antibodies included anti-BRN3A (1:100 dilution, MAB1585; EMD Millipore, Billerica, MA, USA), anti-SOX9 (1:500 dilution, AB5535; EMD Millipore), anti-PKCα (1:100 dilution, sc-208; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Calretinin (1:50 dilution, AF5065; R&D Systems, Minneapolis, MN, USA), and anti-heparin sulfate (1:100 dilution, MAB2040; EMD Millipore). After incubation, sections were washed thoroughly in PBS and then incubated in the appropriate secondary antibody conjugated to either Alexa-488 or FITC. All secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA) and used at a dilution of 1:1000 in PBS, 2% BSA, and 0.1% Triton X-100 buffer. Secondary antibodies were applied for 2 hours at 22°C, or overnight at 4°C, followed by washing in PBS. Sections were then incubated in water containing 300 ng/mL 4′,6-diamindine-2′-phenylindole dihydrochloride (DAPI; ThermoFisher Scientific) for 5 minutes at 22°C, followed by washing and mounting with Immu-Mount (ThermoFisher Scientific). Sections were imaged and digitally photographed on a Nikon Eclipse Ti inverted confocal microscope (Nikon, Melville, NY, USA) configured with an Andor Laser Combiner (Andor, Belfast, Northern Ireland). Emission band pass filters used for imaging were tdTomato (589–625 nm) and Alexa-488/FITC (510–540 nm). Individual fields were imaged as Nyquist Z-stacks of 10 to 15 steps (0.22 μm each) and then used to construct three-dimensional images using IMARIS 7.7 image analysis software (Bitplane, Concord, MA, USA).