Ix51 uorescence microscope

Samples were depara nated using standard protocols. Heat induced epitope retrieval was performed either with 10 mM sodium citrate, 0.05% Tween-20, pH 6 or 10 mM Tris (Table S1, Additional le 1) in preboiled buffer for 30 minutes at room temperature (RT). After blocking with 10% normal donkey serum (Merck Millipore) and 5% bovine serum albumin (Sigma-Aldrich) for 1 hour at 37°C, samples were incubated overnight with primary antibodies at 4°C, and secondary antibodies for 1 hour at RT (Table S1, Additional le 1). Prior to mounting with ProLong Gold containing DAPI (Thermo Fisher Scienti c), lipofuscin auto uorescence was quenched with TrueBlack (Biotium) according to manufacturer's instructions. Sections not incubated with primary antibodies acted as controls. Images were taken using Olympus IX51 uorescence microscope (Olympus).

HUVECs were plated in 24-well platesre-coated with Matrigel (Costar Corning) and pre-treatedith CFDA-SE (MedChemExpress, 5 µM) for 10 min at room temperature. Next, HUVECs were cultured in condition medium containing 50% ECM complete medium (Sciencell) and 50% GSC cell supernatants (volume ratio=1:1). Six hours later, cells were imaged using Olympus IX51 uorescence microscope (Olympus, Tokyo, Japan) and angiogenesis patterns (tube lengths) were analyzed using an Olympus Cellsens 1.5 software (Olympus).
In vitro 3D culture Cells at the logarithmic growth phase were collected and re-suspended in the complete medium containing 2.5% Matrigel (volume ratio, Costar Corning) at a density of 10 4 cells/ml. Next, cells (200 µl) were added into 96-well plates pre-coated with agarose. After low-speed centrifugation (1000 ×g, 10 min, 4˚C), cells were cultured at 37°C in a 5% CO 2 incubator. Medium was replaced with fresh medium every other day. Next, these 3D multiple cell tumor spheres were imaged using a microscope after 7 days.