Retinal Immunohistochemistry for GFP Labeling

To estimate the cell density and specificity of GFP labelling in the whole retina, immunohistochemistry was performed on retinal flat-mounts. Five weeks post-intravitreal injection animals were sacrificed with an overdose of sodium pentobarbital by intraperitoneal injection. The cornea and lens were removed and the eye-cups fixed in 4% paraformaldehyde for 2–3 hours. Retinas were washed in 1 × phosphate-buffered saline (PBS) for 10 min and incubated in blocking buffer (10% normal donkey serum, 0.3% Triton X-100) overnight at 4 °C. Retinas were incubated for 6 days at 4 °C in primary antibodies against RNA binding protein with multiple splicing^{43 (link)–45 (link)} (RBPMS, 1:1000 guinea pig anti-RBPMS, gift from Dr. Nicholas Brecha) and choline acetyltransferase (ChAT, 1:100 goat anti-ChAT, Millipore, Billerica, MA, USA) to identify RGCs and cholinergic amacrine cells, respectively. Retinas were then washed in PBS and incubated overnight at 4 °C in Alexa Fluor 488 (1:400 Alexa Fluor^® 488 conjugated rabbit anti-GFP, Molecular Probes, Eugene, OR, USA), Cy3 (1:1000, Cy3 conjugated donkey anti-guinea pig (Jackson Immuno Research Laboratories Inc., West Grove, PA, USA) and Alexa Fluor^® 633 (1:1000 Alexa Fluor^® 633 conjugated donkey anti-goat, Molecular Probes). Retinas were rinsed in PBS, mounted with anti-fade fluorescent mounting medium and coverslipped.