Cm1950 cryostat

For immunohistochemistry of brain cryosections, mice at post-natal day (PND) 14 and PND 60 from all experimental groups were euthanized with carbon dioxide and their brains were fixed overnight at room temperature in 4% paraformaldehyde. The brains were saturated with ascending amount of sucrose (10% – 20% – 30%, 12 hours each step), snap frozen in optimal cutting temperature (OCT) media and kept at −80°C until they were sectioned. Brains were cut using Leica CM1950 cryostat and mounted on positive charged slides (Fischer Scientific). For immunohistochemical staining, the slides were washed with PBS, which was followed by incubation in 1% hydrogen peroxide for 30 minutes to inactivate the endogenous peroxidase, and then incubated in PBS solution containing 0.05% Triton X-100 and 5% normal goat serum (Invitrogen, Carlsbad, CA) for 30 minutes. The brain tissue was incubated with the following antibodies: rabbit anti-Iba1 for microglia (Wako Chemicals, Richmond, VA), mouse anti-GFAP for astrocytes (Sigma-Aldrich, St Louis, MO), anti-NeuN for neurons (EMD Millipore, Billerica, MA), anti-Myelin basic protein (Abcam, Cambridge, MA). The slides were mounted with Prolong gold antifade reagent as mounting medium (Invitrogen, Carlsbad, CA) and viewed using a confocal Zeiss LSM-510 or Zeiss Axioplan 2 microscope.