Dextran tmr solution

A bolus of 100 μl of a 10 mg ml^–1 70 kDa dextran-TMR solution (Life Technologies) was injected via the tail vein into tumour-bearing SHH-MB mice at advanced symptomatic stages, as previously described^{6 (link)}. Brains were then removed 2 h later without perfusion, fixed overnight in 4% paraformaldehyde, embedded in optimal cutting temperature compound (O.C.T.) and then sections prepared at a thickness of 12 μm. Immunofluorescent staining of tissue sections was performed using antibodies against CD31 and P-selectin with appropriate secondary antibodies, counterstained with DAPI to visualize nuclei and then coverslipped using Fluoro-Gel mounting medium as described below. Detection of TMR-dextran in the context of P-selectin and CD31 immunostaining was imaged using a fluorescent microscope (Zeiss Axioobserver), and TIFF images postprocessed using Adobe Photoshop CS6.