Fluoromount mounting medium

The following protocol was optimised on bEnd.3 cells then used. Cells were grown on cover slips in six well cell culture plates (CELLSTAR, Indiana, USA), and fixed with 4% paraformaldehyde for 6 min cell were washed with PBS three times and permeabilized with 0.2% (v/v) Tween-20 in PBS for another 6 min. After washing with PBS X3 and 1% (w/v) bovine serum albumin three times, cells were stained for anti-cadheren 5 (CD144), anti-CD109 (BD Pharmingen) and anti-protein disulfide isomerise (PDI) (Sigma) antibodies. The corresponding secondary antibodies were labelled with Alexa Fluor 488 (Invitrogen) and nuclei were stained with DAPI (Invitrogen). Slides were then mounted with Fluoromount mounting medium (DAKO) and examined with fluorescent microscope (Leica, Microsystems, Germany). Cells were stained at 6, 24, 48 and 72 h post irradiation. Control cells were also stained at each time point.

Experiments started 1 day after seeding of the GBM cell lines at a density of 5,000 cells/12 mm cover slip. The cells were fixed with 4% paraformaldehyde for 10 min, followed by permeabilization with 0.1% Tween (VWR, 663684B, USA) in PBS for 60 min. Additionally, unspecific binding sites were blocked with goat-serum (Sigma-Aldrich, G9023, 10% in PBS) and bovine serum (Sigma-Aldrich, B9433, 3% in PBS) adding 0.3 M glycine (Biomol, 04943, Germany). After washing with PBS, cells were incubated with rabbit anti-VEGF-R2 antibody overnight (Abcam, ab39638, United Kingdom, 1:300 in PBS), followed by incubation with AlexaFluor 488 anti-rabbit IgG (Molecular Probes, A-11008, USA, 1:250 in PBS) for 75 min and then subsequently treated with rhodamine-phalloidin for 30 min (Sigma-Aldrich, P1951, 1:20 in PBS). Further bisBenzimide H 33342 trihydrochloride (Hoechst, Sigma-Aldrich, B2261, 1:1,000 in PBS, 20 min) was applied to counterstain the cell nuclei. Finally, the cover slips were mounted on microscope slides with fluoromount mounting medium (Dako, S3023, Germany).