One day after FG labeling, the animals were deeply anesthetized with intramuscular injections of 0.8 mL/kg of a cocktail containing ketamine (100 mg/mL), 2.5 mL xylazine (20 mg/mL), 1.0 mL acepromazine (10 mg/mL), and 1.5 mL normal saline and transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer. The eyes were enucleated, immersed in fixative for 1 hour, bisected, and postfixed for 3 hours. The eye cups were incubated with 30% sucrose at 4°C overnight and embedded in OCT compound (Sakura Finetec, Torrance, CA). Ten-micrometer-thick sections were obtained along the vertical meridian through the optic nerve head. The sections were washed three times with PBS in triton-X (T-PBS) for 5 minutes and incubated with blocking buffer (T-PBS containing 5% bovine serum albumin) at room temperature for 30 minutes. After blocking, they were incubated overnight at 4°C with primary antibodies against RBPMS and Thy-1 (1:200, mouse; Chemicon, Temecula, CA), 200 kDa neurofilament (NF-H; 1:500, mouse; Chemicon) or III β-tubulin (TUJ1, 1:200, mouse; Covance, Emeryville, CA). Thy-1 and NF-H are RGC markers and III β-tubulin is a neuronal marker used to distinguish RGCs from displaced amacrine cells in the GCL. The sections were incubated with FITC-conjugated anti-rabbit secondary antibody and rhodamine-conjugated anti-mouse antibody at room temperature for 1 hour and then mounted (Gel/Mount; Biomeda, Foster City, CA) antifade mounting solution. Photomicrographs of the retinal sections were taken with a fluorescence microscope (LSM510; Carl Zeiss Meditec). Negative control sections were incubated without primary antibodies. At least three sections from each eye and four eyes were included for quantitative analysis. For each retinal section, the number of cells in the GCL labeled by FG with immunoreactivity of (1) RBPMS and NF/Thy-1/III β-tubulin, (2) RBPMS but no NF/Thy-1/III β-tubulin, (3) NF/Thy-1/III β-tubulin but no RBPMS were counted.