Intracranial Inoculation Is More Potent Than Intranasal Inoculation for Inducing Optic Neuritis in the Mouse Hepatitis Virus-Induced Model of Multiple Sclerosis

Singh, Manmeet; Khan, Reas S.; Dine, Kimberly; Das Sarma, Jayasri; Shindler, Kenneth S.

RGC immunolabeling and counting was performed as in prior studies (Khan et al., 2017) . Briefly, eyes removed at the time of sacrifice were fixed with 4% PFA. Retinas were isolated and whole-mounted on glass slides, washed several times in PBS, permeabilized at −70 • C in 0.5% Triton X 100 solution, then thawed and washed again in 0.5% Triton X 100. Retinas were incubated overnight with goat anti-Brn3a (RGC marker) antibody (Santa Cruz Biotechnology) diluted 1:100 in blocking buffer (PBS containing 2% bovine serum albumin and 2% Triton X 100). After washing in PBS, retinas were incubated for 1 h with alexa fluor-488 anti-goat secondary antibody diluted 1:500. Retinas were then washed and mounted with vectashield mounting medium for fluorescence. Photographs of RGCs were taken in 12 standardized fields at 1/6, 3/6, and 5/6 of the retinal radius from the center of the retina in four quadrants at 40X magnification. RGCs were counted in each field by a blinded investigator using Image-Pro Plus 5.0 (Media Cybernetics, Silver Spring, MD) software.