Ultrastructural Analysis of Retina Samples

Mice were anesthetized with ketamine/xylazine (100 mg kg⁻¹ and 20 mg kg⁻¹). Live animals were perfused via the aorta with 10 ml of sodium cacodylate buffer (0.1 M, pH 7.4) followed by 10 ml of 1⁄2 Karnovsky’s fixative in 0.1 M sodium cacodylate buffer (Electron Microscopy Sciences). Eyes were enucleated and the anterior segment removed. The eyecups were dehydrated and embedded in tEPON-812 epoxy resin. Semithin Sects. (1 μm) were stained with 1% toluidine blue in 1% sodium tetraborate aqueous solution for light microscopy. Ultrathin Sects. (80 nm) were cut from each sample block using a Leica EM UC7 ultramicrotome (Leica Microsystems) and stained with 2.5% aqueous gadolinium triacetate hydrate and Sato’s lead citrate stains using a modified Hiraoka grid staining system [51 (link)]. Grids were imaged using an FEI Tecnai G2 Spirit transmission electron microscope (FEI Company) at 80 kV interfaced with an AMT XR41 digital CCD camera (Advanced Microscopy Techniques) for digital TIFF file image acquisition. TEM imaging of retina samples was assessed, and digital images were captured at 2 k × 2 k pixel, 16-bit resolution.