Optic Nerve Fixation and Sectioning

Optic nerves were fixed in the skull for 24 h in 4% PFA in DPBS at 4 °C and then processed as described previously^{20 (link),102 (link)–104 (link)}. Briefly, nerves were dissected and fixed overnight in 2.5% glutaraldehyde and 2% PFA in 0.1 M sodium cacodylate buffer pH 7.4, rinsed in 0.1 M sodium cacodylate buffer, and post-fixed with 1% osmium tetroxide for 1 h. A series of 40-min incubations in graded acetone was followed by infiltration overnight at 4 °C with 33%, 66%, and 100% resin (Eponate 12; Ted Pella, Redding, CA, USA) diluted in propylene oxide, and by embedding in 100% resin. Staining with 1% PPD was performed on 1-μm cross sections, followed by mounting with Permount.