Mouse anti flk 1

For cytospins, C6 glioma cells in endothelial differentiation medium were seeded on coverslips and cultured for 24 h in hypoxia. Tumors were collected and then divided for frozen section at 5μm thickness. Sections were fixed in 4% paraformaldehyde and blocked with 10% goat serum. The primary antibodies used in this study were as follows: rabbit anti-von Willebrand factor (vWF) (Millipore, Bedford, MA), rabbit anti-CD34, mouse anti-CD31, mouse anti-Flk1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), and goat anti-Notch1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA). The secondary antibodies used were as follows (all from Invitrogen, Grand Island, NY): Alexa Fluor 555-donkey anti-rabbit IgG, Alexa Fluor 568-rabbit anti-mouse IgG, Alexa Fluor 568-rabbit anti-goat IgG, Alexa Fluor 647-rabbit anti-mouse IgG, Alexa Fluor 647-donkey anti-rabbit IgG. Antibodies were diluted in antibody diluent (Beyotime, Jiangsu, China), and incubations were done at room temperature. The images were captured by confocal laser scanning microscopy (Leica, Heerbrugg, Switzerland), and the obtained images were processed by Adobe Photoshop 7.0 (Adobe System Inc., San Jose, CA).

For detection of protein-protein interactions, in situ PLA was performed. The components used (Sigma) were as follows: anti-mouse PLA plus probe, anti-rabbit PLA minus probe and Detection Reagents Orange. HUVEC or U87MG cells were grown on μ-Chamber 12 well on glass slides (Ibidi©, Martinsried, Germany). After reaching 80% confluence or after appropriate treatment of cells, the assay was performed according to the manufacturer’s instructions. Briefly, after fixation and blocking, cells were incubated with the primary antibodies: mouse anti-VEGF (1:250), rabbit anti-VEGF (1:250), rabbit anti-Flk-1 (1:250), mouse anti-Flk-1 (1:250), mouse anti-NCL (1:50), goat anti-RPTPβ/ζ (1:250) (all from Santa Cruz Biotechnology Inc.), mouse anti-α_νβ₃ (1:500; Merck Millipore), mouse anti-PTN (1:500; Abnova, Heidelberg, Germany) and mouse anti-RPTPβ/ζ (1:250; BD Biosciences). Subsequently, cells were incubated with secondary antibodies conjugated with oligonucleotides. After hybridization and ligation of the oligonucleotides, the DNA was amplified. A detection mixture detected the amplicons, resulting in red fluorescence signals. Nuclei were counterstained with Draq5; cells were mounted with Mowiol 4–88 and visualized at room temperature with Leica SP5 confocal microscope.

Cells were lysed with RIPA buffer, as previously described [8 (link)]. Three mg of total protein were incubated with primary antibody for 16 h at 4°C under continuous agitation. The primary antibodies used were: mouse anti-VEGF (3 μg), mouse anti-Flk-1 (3 μg), goat anti-RPTPβ/ζ (3 μg), goat anti-PTN (3 μg) (Santa Cruz Biotechnology Inc.) and goat anti-β₃ (1.5 μg; Merck Millipore). Protein A- and protein G-agarose beads (Merck Millipore) were added, samples were further incubated for 2 h at 4°C, and beads with bound proteins were collected by centrifugation (5,000 g for 5 min at 4°C) and washed twice with ice-cold PBS pH 7.4. Immunoprecipitated proteins were resuspended in SDS loading buffer and analyzed by Western blot.