Luxol Fast Blue Staining of Frozen Sections

Mice were intracardially perfused with 1× PBS followed by 4% PFA/PBS, post-fixed for 2–24 hours, then cryoprotected overnight in 20% sucrose. Fixed spinal cord tissue was embedded and cryosectioned at 20 µm sections. Sections were then rinsed in water followed by 35% and 70% EtOH, then incubated in luxol fast blue solution overnight at 55-60°C. Excess stain was rinsed with 95% EtOH followed by water, then destaining was performed in 0.05% lithium carbonate solution before additional 70% and 100% EtOH rinsing followed by xylene immersion and coverslipping.