Vibrating microtome

Immediately after the last imaging session, mice were sacrificed and perfused with formalin, and their brains were extracted. The tissue was immediately immersed in formalin. Coronal sections (30 μm) were generated using a vibrating microtome (Fisher). For nestin, GFAP, and TUJ-1 staining, sections were incubated for 1 hour in a blocking solution (0.3% bovine serum albumin, 8% goat serum, and 0.3% Triton X-100) at room temperature, followed by incubation at 4°C overnight with the following primary antibodies diluted in blocking solution: (i) anti-human nestin (Millipore), (ii) anti-GFAP (Millipore), (iii) anti-TRAIL (Prosci), and (iv) anti–TUJ-1 (Sigma). Sections were washed three times with PBS, incubated in the appropriate secondary antibody, and visualized using a confocal microscope (Olympus).

Immediately after the last imaging session, mice were sacrificed, perfused with formalin, and brains extracted. The tissue was immediately immersed in formalin. 30 μm coronal sections were generated using a vibrating microtome (Fisher). Sections were washed three times with PBS and visualized using a confocal microscope (Olympus). In a subset of mice, brains were isolated and incubated with or without D-luciferin, and ex vivo whole-brain bioluminescent and fluorescent imaging was performed using the IVIS^® Kinetic system.

Mice with resected tumors or mice with resected tumors and implanted with bENS carrying hMSCs were perfused with formalin and brains were removed and sectioned. The tissue was immediately immersed in formalin. 30 μm coronal sections were generated using a vibrating microtome (Fisher). Sections were washed three times with PBS and nuclei were counterstained with Hoechst 33342 (Life Technologies) and visualized using a confocal microscope (Olympus). In a subset of mice, brains were isolated and fluorescent imaging was performed using an Olympus MVX-10 microscope.