On PD 21, animals were deeply anesthetized via isoflourane anesthesia and rapidly decapitated. Brains were removed, bisected coronally and immersed in rapid-Golgi impregnation solution (FD Rapid Golgistain kit, FD Neurotech) for up to 2 weeks in the dark. After impregnation, brains were flash frozen in isopentane on dry ice and stored at −80°C until sectioned. Brains were sectioned coronally at 100μm on a cryostat. Sections were mounted on slides allowed to dry overnight and processed according to kit directions. Briefly, sections were rinsed in distilled water, fixed in proprietary solutions, rinsed in distilled water, then dehydrated through increasing alcohol concentrations and cleared with Histochoice (AMRESCO). Glass coverslips were attached with Permount and slides were allowed to dry in the dark until analyzed.
