M2 epifluorescence microscope

The detailed procedure has been described previously (Zhang et al., 2019b (link)). Briefly, transverse semi-thin (1 μm) sections of ON were cut on an ultramicrotome (EM UC7, Leica, Wetzlar, Germany) and collected 2 mm distal to the eye (about 1.5 mm distal to the crushed site). The semi-thin sections were stained with 1% para-phenylenediamine (PPD) in methanol: isopropanol (1:1). Four sections of each ON were imaged through a 100× lens of a Zeiss M2 epifluorescence microscope to cover the entire area of the ON without overlap. Two areas of 21.4 μm × 29.1 μm were cropped from the center of each image, and the surviving axons within the designated areas were counted manually. After counting all the images taken from a single nerve, the mean of the surviving axon number was calculated for each ON. The mean of the surviving axon number in the injured ON was compared to that in the contralateral control ON to yield a percentage of axon survival value. The investigators who counted the axons were masked to the treatment of the samples.

ONs were post-fixed in situ with 2% glutaraldehyde and 2% PFA in 0.1 M PBS. Semi-thin (1 µm) cross sections of the ON 2 mm distal to the eye (globe) were collected. The sections were stained with 1% PPD for 0.5 h before washing with methanol: isopropanol (1:1) 3 times × 10 min and then mounted with Fluoromount-G. Four sections of each ON were imaged through a 100× lens of a Zeiss M2 epifluorescence microscope to cover the entire area of the ON without overlap. Two areas of 21.4 µm × 29.1 µm were cropped from the center of each image, and the surviving axons within the designated areas counted manually using ImageJ. After counting all the images taken from a single nerve, the mean of the surviving axon number was calculated for each ON. The mean of the surviving axon number in the injured ON was compared to that in the contralateral control ON to yield a percentage of axon survival value.

For hematoxylin and eosin (H&E) staining, 1-month-old, dispase treated, depigmented and cleared C57BL/6J eyes were washed in PBS three times for 1 h each to wash CUBIC-2. Eyes were then incubated in 50% sucrose/PBS overnight at 4°C before freezing in OCT at -80°C. Serial 50 μm thick cryosections were obtained and stained with H&E. H&E staining was performed by first washing slides in PBS for 5 min before immersing slides in hematoxylin for 1 min followed by a tap water rinse. Slides were then immersed in 50%, 70%, 80%, 90%, and 100% ethanol for 1 min each before being dipped three times in eosin followed by a tap water rinse. Slides were then immersed in 50%, 70%, 90% and 100% ethanol for 1 min each again before being immersed in xylene for 5 min followed by mounting in Permount (Fisher Scientific, #SP15-100) and imaged on a Zeiss M2 epifluorescence microscope at 2.5x and 20x magnification.