Cone arrestin

Eyecups were fixed in 4% PFA in 1X PBS for 20 min at room temperature and then cryoprotected in 30% sucrose in 1X PBS overnight at 4°C. Samples were embedded in OCT (Sakura Finetek), frozen on dry ice, and then sectioned at 16–18 μm on a cryostat (Leica). Slides were blocked with a solution containing 10% normal horse serum and 0.5% Triton X-100 in 1X PBS for 1 hr at room temperature and then stained overnight at 4°C with primary antibodies (Rho4D2 at 1:250 from Dr. Robert Molday, S Opsin at 1:400 from SCBT: sc-14363, Cone Arrestin at 1:1000 from Millipore: AB15282, Otx2 at 1:200 from R and D Systems: BAF1979) diluted in blocking solution. Slides were washed three times with 1X PBS the following day and then incubated in fluorescently labeled secondary antibodies diluted in blocking solution for 2 hr at room temperature, stained with DAPI, washed, and coverslipped using Fluoromount-G (SouthernBiotech). An Olympus FluoView FV1000 was used for confocal microscopy. Cells were counted from single plane confocal images taken at fixed settings. Counts in the central retina were taken adjacent to the optic nerve head (50 μm from the nerve head on the ventral side) and counts in the peripheral retina were taken 50 μm from the peripheral edge on the ventral side.

The following primary antibodies were used: glutamine synthetase (1:250; BD Biosciences), rhodopsin (1:500; Millipore), cone arrestin (1:500; Millipore), PKCα (1:250; BD Biosciences), MPP4 AK4 (1:200; homemade³⁴ (link)), CRB1 AK2 (pH 1.5) (1:200; homemade⁸ (link)), CRB2 (1:200⁸ (link)), p120-catenin (1:100; BD Biosciences), GFAP (1:200; Dako), CD11b (1:100; BD Biosciences); PLVAP (1:200; BD Pharmingen), and VE-cadherin (1:100; BD Biosciences).