Electron Microscopy of Mouse Eye Tissues

Fixation and processing of eyes for TEM was performed following previously published protocols [52 (link)]. In short, anesthetized mice were transcardially perfused with 2% paraformaldehyde, 2% glutaraldehyde, and 0.05% calcium chloride in 50 mM MOPS (pH 7.4). Enucleated eyes were post-fixed for 2 h in the same fixative. Eyecups were dissected, embedded in 2.5% low-melt agarose (Precisionary, Greenville, NC) and sectioned on a Vibratome (VT1200S; Leica, Buffalo Grove, IL). Agarose sections were stained with 1% tannic acid (Electron Microscopy Sciences, Hatfield, PA) and 1% uranyl acetate (Electron Microscopy Sciences), gradually dehydrated with ethanol, and infiltrated and embedded in Spurr’s resin (Electron Microscopy Sciences). 70 nm sections were cut onto copper grids and counterstained with 2% uranyl acetate and 3.5% lead citrate (19 314; Ted Pella, Redding, CA). Samples were imaged on a JEM-1400 electron microscope (JEOL, Peabody, MA) at 60 kV with a digital camera (BioSprint; AMT, Woburn, MA). Image analysis and processing was performed with ImageJ.