Nissl Staining of Brain Sections

The procedure for Nissl-stained sections followed our standard laboratory protocol described previously (Lavenex et al., 2009 (link)). Sections were taken out of the 10% formaldehyde solution, thoroughly washed 2 × 2 hours in 0.1 M phosphate buffer, mounted on gelatin-coated slides from filtered 0.05 M phosphate buffer (pH 7.4), and air-dried overnight at 37°C. Sections were then defatted 2 × 2 hours in a mixture of chloroform/ethanol (1:1, vol.), and rinsed 2 × 2 minutes in 100% ethanol, 2 minutes in 95% ethanol and air-dried overnight at 37°C. Sections were then rehydrated through a graded series of ethanol, 2 minutes in 95% ethanol, 2 minutes in 70% ethanol, 2 minutes in 50% ethanol, dipped in two separate baths of dH₂O, and stained 20 seconds in a 0.25% thionin (Fisher Scientific, Waltham, MA, cat# T-409) solution, dipped in 2 separate baths of dH₂O, 4 minutes in 50% ethanol, 4 minutes in 70% ethanol, 4 minutes in 95% ethanol + glacial acetic acid (1 drop per 100 ml of ethanol), 4 minutes in 95% ethanol, 2 × 4 minutes in 100% ethanol, 3 × 4 minutes in xylene, and coverslipped with DPX (BDH Laboratories, Poole, UK).