Transmission electron microscopy
Littermate Crx-/- and wildtype pups were perfused in 1% formaldehyde and 0.5% glutaraldehyde at various postnatal stages. The eyes were then enucleated, and the cornea and lens were removed. The eye cup was immersed in fixative (1% formaldehyde and 2.5% glutaraldehyde) for 3 to 4 hours at 4°C. The sclera was then partially removed and the retinas were sliced into small pieces and fixed (1% paraformaldehyde and 2.5% glutaraldehyde) overnight at 4°C. These procedures were found optimal for maintaining the structural integrity of the photoreceptor outer segments.
After fixing, the tissue was washed 2X in PBS for thirty minutes per wash. The tissue was then postfixed in a 1% osmium tetroxide/1.5% potassium ferrocyanide mixture for 2 hours at 4°C. Staining was carried out for 30 minutes in 1% uranyl acetate in maleate buffer (pH = 6.0) at room temperature followed by 1% tannic acid in 0.1 M cacodylate buffer (pH = 7.4) for thirty minutes. The specimens were then dehydrated and embedded in Epon/Araldite. Thin sections were stained with uranyl acetate and lead citrate, and examined in a Jeol JEM-1200EX electron microscope.