Mouse anti ανβ3

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 fixed with 4% formaldehyde in phosphate-buffered saline (PBS) pH 7.4 for 10 min and permeabilized with 0.1% Triton in PBS for 15 min. After being washed 3 times with PBS, the cells were blocked with PBS containing 3% BSA and 10% fetal bovine serum (FBS) for 1 h at room temperature. Cells were stained with the following primary antibodies: rabbit anti-VEGF (1:250; Santa Cruz Biotechnology Inc.), mouse anti-RPTPβ/ζ (1:250; BD Biosciences, San Diego, CA, USA), mouse anti-α_νβ₃ (1:500; Merck Millipore, Darmstadt, Germany), and rabbit anti-NCL (1:1,000, Sigma). Cells were then incubated with fluorescent Alexa secondary antibodies (1:500; Molecular Probes, Carlsbad, CA, USA). Nuclei were stained with Draq5 (Biostatus Limited, Leicestershire, UK). Cells were mounted with Mowiol 4–88 (Merck Millipore) and visualized at room temperature with Leica SP5 (X63 objective with a numerical aperture of 1.4) confocal microscope.