Mice were anesthetized and transcardially perfused with PBS/heparin (Hospira, Lake Forest, IL, USA) to clear blood cells from the vasculature. The brains were dissected out and meninges removed and fixed in 4% paraformaldehyde (Boston Bioproducts, Ashland, MA, USA) for 3–4 h at room temperature then cryopreserved in 30% sucrose overnight at 4 °C. Tissue was then placed in OCT Compound (Tissue Tek, Torrance, CA, USA) and frozen at − 20 °C. Twelve to 14 μm sagittal sections were cut using a Leica LM3050S cryostat (Buffalo Grove, IL, USA). The tissue sections were washed with PBS, blocked in 1% BSA (Fisher BioReagents, Fair Lawn, NJ, USA) and 1% normal goat serum (Invitrogen, Carlsbad, CA, USA) with 0.3% Triton X-100 (Sigma) at room temperature for 2 h, stained overnight with the appropriate primary antibodies (Table 1) at 4 °C, washed, stained with appropriate secondary antibodies (Table 1) for 1 h at room temperature, and mounted with Prolong Gold Antifade Reagent (Invitrogen). Images were acquired using either an LSM 700 scanning confocal microscope with Zeiss Imager.Z2 or a Zeiss Observer.Z1 confocal microscope equipped with the Zen Blue acquisition software (Zeiss, Oberkochen, Germany).
