Retinal and Optic Nerve Tissue Preparation

After 14 days, eyes (n = 13/group) and optic nerves (n = 8/group) were explanted. Five retinae were frozen directly at −80 °C for later quantitative real-time PCR (RT-qPCR) analysis. The other organs were prepared for retinal cross-sections (n = 8/group) or longitudinal optic nerve (n = 8/group) sections, as previously described [17 (link)]. The organs were fixed in 4% paraformaldehyde (eyes for 1 h and optic nerves for 2 h, Merck, Burlington, MA, USA), cryo-conserved in 30% sucrose overnight, and frozen embedded in NEG-50 Tissue Tek medium (Thermo Fisher Scientific, Waltham, MA, USA). The retina cross-sections (10 µm) and longitudinal optic nerve sections (4 µm) were attached on glass slides (Superfrost Plus, Thermo Fisher Scientific), fixed in ice-cold acetone, and used for immunohistological staining.