Following euthanasia and enucleation, corneas were punctured and eyes (minimum of four each from TG and WT animals at P30 and P90) placed in ice-cold TEM-fixative (0.1 M cacodylate buffer, pH 7.4, with 2 mM sucrose, 4% paraformaldehyde [PFA], 2.5% glutaraldehyde [GA]) for 3 hours at 4°C, after which time corneas and lenses were removed. Following 30 minutes of additional fixation, four cuts were made to flatten the posterior eyecup, 1 × 2-mm sample pieces were cut from the central retina 250 to 400 μm from the optic nerve head, and fixation continued for 72 hours at 4°C.
Sample pieces then were washed for 1 hour in 0.1 M sodium cacodylate buffer, to remove any residual aldehyde, and incubated in 2% osmium tetraoxide (OsO4) in 0.1 M sodium cacodylate buffer for 1 hour to fix lipids. Brief washing with 0.1 M sodium cacodylate buffer (15 minutes), then with 0.1 M sodium acetate buffer, pH 5.2 (5 minutes), was followed by en bloc staining with 2% uranyl acetate in 0.1 M sodium acetate buffer, pH 5.2 for 1 hour to increase contrast. After further washing with 0.1 M sodium acetate buffer, the sample pieces were dehydrated in an ethanol series (50, 70, 80, 90, 95, and 100%), followed by infiltration with Spurr's resin (Sigma-Aldrich Corp., St. Louis, MO, USA). Sample pieces then were placed into flat embedding molds, oriented parallel to photoreceptor outer segments, and the resin thermally polymerized for 24 hours at 65°C. Ultrathin sections (60 nm) were cut with an ultramicrotome (Leica UC7; Leica Microsystems, Inc., Ontario, Canada), and then stained with 2% uranyl acetate and Reinold's lead citrate. The contrasted sections were imaged under a Hitachi H-7650 transmission electron microscope (Hitach High-Technologies Canada, Inc., Rexdale, ON, Canada) at 80 kV equipped with a 16 mega pixel EMCCD camera (XR111; Advanced Microscopy Techniques, MA, Woburn, USA) and imaging software (AMT version 600; Advanced Microscopy Techniques).