Mouse Eye Ultrastructure Visualization

Fixation and processing of mouse eyes for TEM was performed as described previously (Ding et al., 2015 (link)). Anesthetized mice were transcardially perfused with 2% paraformaldehyde, 2% glutaraldehyde and 0.05% calcium chloride in 50 mM MOPS (pH 7.4) resulting in exsanguination. Enucleated eyes were fixed for an additional 2 h in the same fixation solution at room temperature. Eyecups were dissected from fixed eyes, embedded in 2.5% low-melt agarose (Precisionary, Greenville, NC, USA) and cut into 200 μm thick slices on a Vibratome (VT1200S; Leica, Buffalo Grove, IL, USA). Agarose sections were stained with 1% tannic acid (Electron Microscopy Sciences, Hatfield, PA, USA) and 1% uranyl acetate (Electron Microscopy Sciences), gradually dehydrated with ethanol and infiltrated and embedded in Spurr’s resin (Electron Microscopy Sciences). Seventy nanometer sections were cut, placed on copper grids and counterstained with 2% uranyl acetate and 3.5% lead citrate (19314; Ted Pella, Redding, CA, USA). The samples were imaged on a JEM-1400 electron microscope (JEOL, Peabody, MA, USA) at 60 kV with a digital camera (Orius; Gatan, Pleasanton, CA, USA). Image analysis and processing were performed with ImageJ. For each genotype, over 100 outer segments from at least two mice of randomized sex were analyzed.