Alexa 647 conjugated ctb

Mice were anesthetized with 2% isoflurane before ONC. Optic nerves were exposed intraorbitally and crushed at approximately 0.5–1.0 mm from the posterior pole of the eyeball with fine surgical forceps for 5 seconds^{1 (link),2 (link),28 (link)}. On d12 after ONC, 2 µg of Alexa 647-conjugated CTB (Invitrogen, Carlsbad, CA, USA) was injected into the vitreous body. On d14 after ONC, the animals were perfused with Zamboni’s Fixative (2% paraformaldehyde and 15% picric acid in 0.1 M phosphate buffer). The optic nerve was removed, postfixed and immersed in 30% sucrose overnight at 4 °C. The optic nerve was then embedded in an OCT compound (Sakura, Tokyo, Japan), frozen on dry ice and 14-µm serial cross-sections were prepared using a cryostat and collected on MAS-coated glass slides (Matsunami, Osaka, Japan). To estimate the total number of regenerating axons, axonal outgrowth was quantified by counting CTB-positive axons that crossed a virtual line parallel to the lesion site at 100, 250 and 500 µm distal to the lesion site.