Retinas were harvested at P15, P25, and P60. Eyes were removed from the animal immediately following cervical dislocation and fixed in 4% paraformaldehyde (PFA) in PBS for 20 minutes. Eyes were then removed from the PFA solution and placed in PBS for dissection. An incision was made in the cornea, through which the lens was gently removed. Eyecups were returned to 4% PFA in PBS overnight. Eyecups were sequentially immersed in 10%, 20%, and 30% sucrose in PBS, mounted in optical coherence tomography (OCT) medium (Tissue-Tek; Sakura Finetek, Torrance, CA, USA) and frozen at −80°C. Horizontal 14 to 16 μm cryostat sections were blocked in 10% goat serum in PBS, 1.0% Triton X-100 solution for at least 2 hours, and then incubated with primary antibodies in a solution containing 5% goat serum and 0.1% Triton X-100 in PBS overnight at 4°C. Following three 5-minute washes in PBS, tissue was incubated with secondary antibodies for 1 hour at room temperature.
The following antibodies and stains were used at the noted dilutions for this study: SOX2, rabbit polyclonal (1:2000; Merck Millipore, Billerica, MA, USA); SOX2 mouse monoclonal (1:100; R&D Systems, Minneapolis, MN, USA), cellular retinaldehyde–binding protein (CRALBP, 1:500; Abcam, Cambridge, UK); Glutamine Synthetase (GS, 1:1000; Merck Millipore); β-galactosidase (1:10,000; Molecular Probes, Eugene, OR, USA); SOX9 (1:1000; Merck Millipore); Calretinin (1:500; Merck Millipore); Neurofilament (1:5000; Hybridoma Bank, University of Iowa, Iowa City, IA, USA); glial fibrillary acidic protein (GFAP, 1:500; DAKO, Glostrup Municipality, Denmark); Cleaved Caspase 3 (1:250; Cell Signaling Technology, Inc., Danvers, MA, USA); goat anti-mouse IgG1 (AlexaFluor 488 conjugate, 1:2000), goat anti-rabbit IgG (AlexaFluor 488 conjugate, 1:2000), goat anti-mouse IgG2a (AlexaFluor 546 conjugate, 1:1000), goat anti-rabbit (AlexaFluor 546 conjugate, 1:1000), Hoechst 33258 (1:10000; Invitrogen, Carlsbad, CA, USA). Z-stack images were collected on a confocal scanning microscope (LSM 710; Carl Zeiss Microscopy, LLC, Thornwood, NY, USA), collapsed, and processed using graphic editing software (Adobe Photoshop; Adobe Systems, San Jose, CA, USA).