Vibratome

Mouse brains were fixed with 4% paraformaldehyde, sectioned by vibratome (Leica) or cryostat (Leica) and stained following established protocols^{6 (link)}. For brain slice assays^{6 (link)}, 250 μm thick slices of adult mouse brain were prepared with a vibratome (Leica) and placed on top of 0.8 μm pore membranes (Millipore) in brain slice culture medium (DMEM, complete HBSS, 5% FBS, 1mM L-glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin). 3 × 10⁵ cancer cells were placed on the surface of the slice. After 48 h of incubation, brain slices were fixed with 4% paraformaldehyde, and stained. For immunostaining in chamber slide cultures, cells were fixed with 4% paraformaldehyde and stained. Antibodies used for immunochemical staining are listed in Supplementary Information. Images were acquired with Zeiss Axio Imager Z1 microscope or Leica SP5 upright confocal microscope, and analyzed with ImageJ, Imaris and Metamorph softwares. Antibodies used for immunostaining are listed in Supplementary Information.

All viruses were obtained from the University of North Carolina (UNC) Vector Core. Mice were stereotactically injected with rAAV5-EF1alpha-DIO-eYFP-WPRE (“DIO-eYFP”, UNC Vector Core) into the right mPFC, 1.5 μL total volume (750 nL at 150 nL/min at +0.30–0.32 / +1.70 / –2.25 plus 750 nL at 150 nL/min at +0.30–0.32 / +1.70 / –2.75). Coordinates are given as mm from Bregma (medial-lateral / anterior-posterior / dorsal-ventral). Fluorescently labeled CTB was injected into either the left mPFC (to label IT cells) or the right MD thalamus (to label SC cells). After 4 days, animals were deeply anesthetized with Euthasol and transcardially perfused with 4% paraformaldehyde. Brains were incubated in 4% PFA overnight and then sliced on a Leica vibratome into coronal sections 100 μm thick. Brain slices were mounted onto glass slides and imaged using a confocal microscope. We counted 100 cells per slice in prelimbic and infralimbic cortices in the area that had the maximum overlap between eYFP and the fluorescently labeled CTB.

Fresh tumor tissue was collected at the time of resection. The tumor tissue was mechanically and enzymatically dissociated using a collagenase-based dissociation method as previously reported⁴¹ . Early embryonic hindbrain structures were dissected from the gestational time points E10 and E12. An incision was made between the midbrain and hindbrain boundary, as well as between the prepontine hindbrain and pontine hindbrain, in order to isolate the isthmus, and rhombomeres −1 and −2 at these early time points in development. Late embryonic cerebellar primordia were collected at embryonic time points 14, 16 and 18. All embryonic mouse dissections were performed under a Leica stereoscope with a pair of Moria ultra fine forceps (Fine Science Tools). The tissue was transferred into ice cold Leibovitz’s medium, followed by single cell dissociation with the Papain Dissociation System (Worthington Biochemical Corporation). Postnatal cerebella were dissected from the following time points: day 0, 5, 7 and 14. The central nervous system was fully dissected, then embedded in 2% Low melting point agarose. One mid sagittal slice of 300 um was generated using the Leica vibratome⁴² . Under the stereoscope the cerebellum was isolated from the slice, followed by immediate single cell dissociation as described above.