Wholemount preparations and cryosections of retinas were performed as described22,42. Briefly, whole retinas were incubated for 1-2 hours in blocking buffer (0.4% Triton-X, 4% normal donkey serum in PBS), then incubated for 6 days at 4°C with primary antibodies. Sagittal 80μm sections of cerebellum were obtained with a vibratome (Leica), incubated in blocking buffer, and with primary antibodies for 2 days at 4°C. Following washing, retinas and brain sections were incubated for 3 hours at room temperature with Alexa-conjugated secondary antibodies (Invitrogen or Jackson ImmunoResearch). Whole retinas were flattened with photoreceptor side down onto nitrocellulose filters. Retina flatmounts and brain sections were mounted onto glass slides, coverslipped with Vectashield (Vector) or Fluoromount G (Southern Biotech), and imaged on an Olympus FV1000 scanning confocal microscope. Antibodies used were as follows: chick and rabbit anti-GFP (Aves and Millipore); rabbit anti-DsRed (Clontech); goat anti-choline acetyltransferase (Millipore); guinea pig anti-vGluT3 (Millipore); rabbit anti-Calbindin (Swant); mouse anti-syntaxin HPC1 clone (Sigma); rabbit anti-cleaved caspase3 (Cell Signaling Technology). Nuclei were labeled with DAPI, Po-pro1, or NeuroTrace Nissl 435/455 (Invitrogen).
