Fluoro-Jade C staining

Neurodegeneration was detected using Fluoro-Jade C (FJC) staining as previously described [6]. After deparaffinization and rehydration, the brain sections were successively incubated with 80% alcohol containing 1% NaOH for 5 min, 70% alcohol for 2 min, 0.06% potassium permanganate for 10 min, and 0.0001% FJC (AG325, Millipore, Germany) working solution for 30 min. Next, sections were washed and dried in an incubator for 10 min and cleared in xylene and coverslipped with a non-aqueous, low-fluorescence, styrene-based mounting medium (Sigma-Aldrich, USA). Microscopy of the stained brain sections was performed by an experienced pathologist who was blinded to the experimental condition.