Animals were anesthetized with chloral hydrate (0.1 ml of a 15% solution, i.p.), and perfused transcardially with ice-cold 1% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4) for 1 minute followed by 4% paraformaldehyde in 0.1 M PBS with 0.125% glutaraldehyde for 12 minutes. The brains were carefully removed from the skull and postfixed overnight at 4°C in 4% paraformaldehyde in PBS with 0.125% glutaraldehyde. The brains were then transferred to PBS, sectioned at 200 μm on a Vibratome (Leica VT1000S, Bannockburn, IL, USA), and stored at 4°C in PBS until ready for use.
