Brain Tissue Preparation and Staining

Mice were transcardially perfused with PBS then 4% formaldehyde. Brains were removed and post-fixed in 4% formaldehyde overnight, then sliced with a Pelco easiSlicer vibratome (Ted Pella Inc., Redding, CA) into 50 μm slices. Slices were subjected to the ‘Brain BLAQ’ protocol (82 (link)) before staining, then blocked in PBST with 5% BSA overnight. For electrode implanted animals, slices were stained with GS-IB₄-Alexa Fluor 488 Conjugate (1 μg/mL; Invitrogen, Carlsbad, CA #I21411), propidium iodide (1 μg/mL; Molecular Probes, Eugene, OR), and DAPI (0.25 μg/mL; Invitrogen) in blocking solution overnight (Figure 4A). For viral injected animals, the virally expressed GFP or YFP was enhanced using the rabbit anti-GFP Tag Polyclonal Antibody-Alexa Fluor 488 (Invitrogen #A21311). In the viral rescue experiment (Figure 5A–B), slices were also stained with a goat anti-ChAT primary antibody (1:500; #AB144P, Millipore, Burlington, MA), and Donkey anti-Goat secondary antibody-Alexa Fluor 594 (1:1000, #A-11058, Invitrogen). Slices were washed in PBST four times over a 16–24 hour period, then 3 times over a one hour period in PBS. Slices were mounted in Fluoromount (Sigma-Aldrich, St. Louis, MO) and imaged using an AxioZoom.V10 microscope (Zeiss).