Animals were transcardially perfused with saline (2min) followed by 4% paraformaldehyde (10min). Brains were post-fixed overnight (4% PFA) followed by 30% sucrose (two days) and then flash frozen. Luxol Blue and Crestyl Violet staining were performed as previously described55. Briefly, free floating sections (50um) were washed in 0.1mPBS (3×10min), 70% ethanol (2×5min) and incubated in 70% ethanol overnight (room temperature). Sections were transferred to prewarmed Luxol Fast Blue solution (0.1% Luxol Fast Blue Solvent 38 (Sigma) dissolved in 96% ethanol and 10% acetic acid) and incubated overnight at 56C. Sections were washed sequentially (3min) in water, 0.1M PBS, and lithium carbonate solution (0.05% lithium carbonate in water). Sections were differentiated in 70% ethanol (3min), washed twice (5min) in 0.1M PBS, mounted and dried overnight. The next day sections were crestyl violet stained: defatting (30min 50% chloroform/50% ethanol), dehydrations (100%, 95%, 70% and 50% ethanol sequentially), rinsed (water), Cresyl Violet stained (6 dips), rehydrated (50%, 70%, 95%, 100% ethanol), cleared (Hemo-De) and coversliped.
