Optic Nerve Immunohistochemistry Post-Injury

Optic nerves were harvested 6 and 24 hours post ONC and SI-TON. Optic nerves were fixed, sectioned, permeabilized, and blocked as previously descrived.^{19 (link)} Sections were incubated with primary antibodies (mouse anti-GFAP [anti–glial fibrillary acidic protein], 1:400, Sigma; rat anti-CD45, 1:200, eBioscience, San Diego, CA, USA; goat anti-TNF, 1:50, Santa Cruz, Dallas, TX, USA) in blocking solution overnight at 4°C. After washing in PBS three times, sections were incubated with species-specific fluorescent secondary antibodies. Control sections were incubated with secondary antibody alone. Finally, sections were coverslipped with Vecta shield (Vector, Burlingame, CA, USA) fluorescent mounting medium containing DAPI. Imaging was performed with Leica TSL AOBS SP5 confocal microscope (Leica Microsystems, Exton, PA, USA).