Goat anti mouse 660

Tumor pieces were fixed with 4% PFA, embedded in paraffin and cut into 3 µm sections. Tumor sections were immersed in a 10% blocking solution of the specific serum and then incubated (overnight, 4°C) in solutions containing the following primary antibodies: goat anti-mouse CD105 (R&D Systems, ref. AF1320), mouse anti-human vimentin (Santa Cruz Biotechnology, ref. sc-6260) and rabbit anti-VEGF (Abcam, ref. ab52917). Then, Alexa Fluor-conjugated secondary antibodies were used for 1 h (donkey anti-goat 568 and goat anti-mouse 660, LifeTechnologies, ref. A-11057 and A-21055, respectively; and donkey anti-rabbit 647, Abcam ref. ab150075), and the nuclei were counterstained with DAPI. Coverslips were mounted using Fluor-Save™ reagent (Calcibiochem, ref. 345789). Five random areas of each tumor were imaged using a Leica Microsystems THUNDER 3D Assay Imager epifluorescence microscope. Immunofluorescence images were quantified with the open-source software FiJi version 2.3.051. The background was subtracted from CD105 and VEGFA raw intensity images, then the integrated intensity density of each image was multiplied by the number of pixels in the image and divided by the number of cells that were stained by DAPI.

Immunofluorescence staining for mouse albumin was performed to determine the vascular integrity of the brain. Brain sections obtained from GBM27 and GBM38 hCSCs and PBS control xenotransplants were incubated with a 5% blocking solution of the specific serum, and then incubated (overnight, 4°C) in solutions containing the following primary antibodies: goat anti-mouse CD105 (R&D Systems), goat anti-mouse albumin (Santa Cruz Biotechnology), and mouse anti-human vimentin (Santa Cruz Biotechnology). Then, Alexa Fluor-conjugated secondary antibodies were used for 1 h (donkey anti-goat 568, rabbit anti-goat 488, and goat anti-mouse 660; Life Technologies, USA), and then nuclei were counterstained with DAPI and coverslips were mounted using FluorSave™ reagent (Millipore). Fluorescence was examined under a Leica TCS SP5 inverted confocal microscope.