Mouse Brain Slice Assay for Cancer Cell Invasion

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.