Brain Tissue Preparation and Staining for GFP and CD31

Three days after intracarotid injections, mice were sacrificed and their brains were harvested and processed for frozen or paraffin sections, as previously described.^{16 (link)} Staining with H & E or DAPI (FluoroPure grade, Invitrogen) was performed to visualize the tumor. The GFP-labeled cells were visualized in frozen sections using fluorescence microscopy and in paraffin sections after deparaffinization and antigen retrieval with 1:1000 rabbit anti-GFP primary antibody (Novus) and 1:200 Donkey anti-rabbit green secondary antibody (Invitrogen). Detection of endothelial cells was performed with 1:250 goat anti-CD31 primary antibody (R&D) and 1:200 donkey anti-goat red secondary antibody (Invitrogen). For U87 xenografts, specimens were fixed in 10% formalin for 48 hours, placed in 30% sucrose until they sank, and then embedded in optimal cutting temperature medium and frozen.