At 6-8 weeks postinjection, the eyes were harvested and fixed in 4% paraformaldehyde for 1 hour at room temperature. Whole mounts were prepared by making a circular incision around the ora serrata, removing the cornea and gently manipulating the eye cup to remove the neural retina. For retinal cross-sections, whole mounts were embedded in agarose and sectioned transversely using a vibratome (Leica Microsystems) at medium speed, maximum vibration, and 100 μm thickness. Whole mounts and sections were mounted on slides using VECTASHIELD antifade mounting medium (Vector Laboratories) containing DAPI to stain cell nuclei and examined under confocal microscopy to confirm expression of the opsin-YFP fusion proteins. For staining of rhodopsin, mounted sections were permeabilised with 0.2% Triton-X-100 PBS for 20 minutes at room temperature then blocked with 10% donkey serum (D9663, Sigma UK) in 0.2% Triton-X-100 PBS for one hour at room temperature. Primary antibody solution was then applied for 3 hours at room temperature (1 : 200 rabbit anti-human rhodopsin, Abcam ab112576 in blocking buffer). Sections were rinsed with 0.05% Tween20 PBS four times for 10 minutes each then incubated with secondary antibody solution for 2 hours at room temperature (1 : 200 AlexaFluor donkey anti-rabbit 488 in 2.5% donkey serum, 0.2% Triton-X-100 PBS). Slides were rinsed four times in 0.05% Tween20 PBS then once with water before being DAPI mounted as described above.
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