CD-1 mice (Charles River) were killed with an overdose of isoflurane and transcardially perfused with aCSF (see Single Cell Dissociation, Brain). Brains were dissected out, snap frozen in OCT on a bath of isopentane with dry ice and stored at –80°C. Fresh frozen sagittal whole-brain sections (including the olfactory bulb, SVZ, hippocampus and cerebellum) of 10 μm thickness were cryosectioned and stored at –80°C. Sections were thawed just prior to staining and fixed with 4% PFA for 15 min followed by rinsing in PBS. RNAscope in situ hybridizations were performed according to the manufacturer’s instructions, using the RNAscope Multiplex Fluorescent kit (Advanced Cell Diagnostics) for fresh frozen tissue, as previously described (Hochgerner et al., 2018). A 10 min treatment in SDS (4% in 200 mM sodium borate) was added in the protocol after the Protease IV incubation. Following probes with suitable combinations were used (indicated with gene target name for mouse and respective channel, all Advanced Cell Diagnostics): Mfge8 (Ch1), Agt (Ch2), Aqp4 (Ch3), Slc6a9 (Ch1), Slc6a11 (Ch3), Islr (Ch1) and Gdf10 (Ch2). All sections were mounted with Prolong Diamond Antifade Mountant (Thermo Fisher Scientific). Imaging was carried out on a Nikon Ti-E epifluorescence microscope at 10X magnification.
