Quantifying Intact Retinal Ganglion Cell Axons

Neurofilament staining in optic nerve sections was done to quantify the area of intact RGC axons using a previously published protocol [11 (link), 38 (link)]. Briefly, optic nerves were isolated following sacrifice after 42 days, fixed in 4% paraformaldehyde, and embedded in paraffin. 5 μm longitudinal paraffin sections of the optic nerve were deparaffinized, rehydrated, and then permeabilized with 0.5% tween-20 in PBS. Blocking reagent (Vector Laboratories, Burlingame, CA, USA) was used to reduce nonspecific binding. Specimens were then incubated in rabbit anti-neurofilament antibody 1 : 100 (Abcam, Cambridge, MA, USA) at 4°C overnight and then washed three times with PBS and incubated with anti-rabbit secondary antibody (Vectastain Elite ABC Rabbit kit) for 30 min at 37°C. Avidin/Biotin Complex detection was performed using the Vectastain Elite ABC kit and DAB (diaminobenzidine) substrate kit (Vector Laboratories) according to the manufacturer's instructions. Photographs of three fields/nerve (one each at the distal, central, and proximal regions of the longitudinal optic nerve section) at 20x magnification were taken by a blinded investigator. Neurofilament staining was quantified by calculating the optical density using ImageJ software (http://nih.gov).