Fisherbrand superfrost

Enucleated eyes were briefly incubated in 4% PFA and replaced with phosphate-buffered saline (PBS) before the cornea, anterior segment, and lens were resected under a stereo dissection microscope (SMZ800; Nikon, Tokyo, Japan). Following removal from the eye cup, retinae were incubated in 2% PFA for 15 minutes at room temperature before being rinsed three times in PBS, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) for 15 minutes and subsequently blocked in PBS containing 5% v/v normal goat serum (R&D Systems, Minneapolis, MN) and 2.5% v/v 10% bovine serum albumin (Sigma-Aldrich) for 1 hour. Samples were incubated with a primary GFP polyclonal antibody (1:500 dilution, A-6455; Invitrogen, Carlsbad, CA) at 4°C overnight. After the retinae were rinsed with wash buffer (PBS containing 0.001% Triton X-100) four times, samples were incubated with secondary antibodies to rabbit IgG pre-labeled with Alexa Fluor 488 (1:2000 dilution, A-21206; Invitrogen). Retinae were finally rinsed with wash buffer four times, mounted on microscope slides (Fisherbrand Superfrost; Thermo Fisher Scientific) before being imaged using a confocal microscope (Nikon Eclipse 80i) with a 20× objective and 488-nm and 561-nm lasers (Coherent, Santa Clara, CA). The z-stacks covering the full thickness of the retina were recorded, and the images were processed using ImageJ.