Immunocytochemistry

Before enucleation, the superior pole of the cornea was marked by cauterization and the cornea, lens, and vitreous were subsequently removed. The remaining eye cups were placed in 4% paraformaldehyde in PBS for 15 min and rinsed 3 times for 10 min in PBS. The eye cups were cryoprotected in 30% sucrose in PBS for 2 h, placed in Tissue-Teck® O.C.T. compound (Sakura Finetek, USA) and quickly frozen in liquid N₂. The frozen blocks were sectioned at 10 μm in a cryostat (CM 3050 S, Leica Microsystems) and stored in −80 °C. Prior to antibody incubation, the sections were equilibrated to room temperature (RT) for 15 min. For GNAT1 staining using the TF-15 mouse monoclonal antibody, epitope retrieval was performed: sections were treated for 2 min RT with 0.02 mg/ml proteinase K in blocking buffer (2% bovine serum albumin, 2% goat serum, 0.3% Triton X-100 in 1X PBS) and heated to 65 °C for 10 s followed by five rinses with PBS. Blocking buffer was then applied to all sections for 1 h. Sections were either incubated with the rabbit antibody against ARR1 (C10C10 [33, 34] diluted 1:100 in blocking buffer) or TF-15 (CytoSignal, diluted 1:200 in blocking buffer). The sections were rinsed and incubated with a fluorescein-labeled secondary antibody (Vector Laboratories). All sections were then double-stained with the biotinylated antibody against rhodopsin (1D4 [35] diluted 1:300 in blocking buffer). The sections were rinsed and incubated with rhodamine Avidin D (1:100, Vector Laboratories). Images were obtained using a Zeiss AxioPlan2 microscope. Light and dark conditions were imaged using identical exposure times.