Donkey anti rabbit or mouse alexa fluor 488 or 568

Retinal histology and immunohistochemistry was performed at 3 and 7 months after vector injection (n=15 per treatment group). Enucleated eyes were preserved in 4% PFA for 2 hr, cryo-protected in 30% sucrose at 4°C overnight, and embedded in tissue freezing media (Triangle Biomedical, Durham, NC). Ten-micron thick sections were collected on a cryostat (Fisher, Pittsburgh, PA). Slides were rinsed with PBS and incubated at room temperature for 2 hr in normal donkey serum (1:20) in 0.1 M phosphate buffer with 0.5% bovine serum albumin and 0.1% Triton X 100 (PBT). The slides were incubated overnight at 4°C in anti-rhodopsin (1:50, Abcam, Cambridge, MA) or anti-glial fibrillary acidic protein (GFAP, 1:400, Dako, Carpinteria, CA) in PBT, then rinsed with PBS and incubated for 2 hr at room temperature in the appropriate fluorophore-conjugated secondary antibody (donkey anti-rabbit or mouse -Alexa Fluor 488 or 568, Life Technologies, Carlsbad, CA). Slides were rinsed with PBS, mounted in Vectashield Mounting medium with DAPI (Vector Laboratories, Burlingame, CA), and imaged on a Nikon Eclipse epifluorescence microscope (Nikon, Melville, NY). Images were collected at the same magnification, gain, and exposure settings.