Shd 42 15

After performing PS-OCT imaging, the brain slice was transferred onto No. 0 glass-bottom 35 mm culture dishes (P35G-0-20-C; MatTek, Ashland, MA). U251 cells (500,000–800,000) were plated onto the brain slice. Isolectin GS-IB4 (Alexa Fluor 568; Molecular Probes, Eugene, OR) was added to the brain slice to label the vasculature. Isolectin IB4 labels endothelial cells and has been used as a robust marker for vasculature (27 (link)). The cells were cocultured with the brain slice for 4 h before imaging to allow for cell infiltration into the brain slice. Cells typically invade up to 50 μm into the tissue. Before imaging, the brain slice was washed several times using cell culture media. A tissue culture anchor (SHD 42-15; Warner Instruments, Hamden, CT) was placed on top to prevent tissue drift during imaging.

Brain slices were transferred into DMEM/F12 media containing 8% FBS and 1x P/S and stored in an incubator at 37 °C and 5% CO₂. Two hundred thousand cells were plated onto a slice and incubated overnight to allow cells to infiltrate into the slice. Cells typically invade up to 100 μm into the tissue. Isolectin GA-IB4 (Alexa Fluor 568; Molecular Probes, Eugene, OR) was added to the slice to stain the vasculature at least 30 min before imaging. The slice was transferred into a 6-well glass-bottom 35 mm culture dish (P35G-0-20-C; MatTek, Ashland, MA) with fresh media. A tissue culture anchor (SHD 42-15; Warner Instruments, Hamden, CT) was placed on top of the brain slice to prevent it from drifting during imaging.