Rabbit anti vegfr2

Cell cultures were fixed with 2% paraformaldehyde (PFA; Sigma), washed with PBS and blocked with a solution of 3% BSA and 0.1% Tween 20 (Sigma) for 20 min at room temperature to prevent nonspecific binding. Cells were incubated with the primary antibody for 30 min at room temperature followed by overnight incubation at 4 °C. Incubation with the respective secondary antibody was performed at room temperature for 2 h. The antibodies used were rabbit anti-Argonaute-2 (Ago2) (1:200; Cell Signaling, MA, USA), rabbit anti-neuropilin-1 (NRP1) (1:200; BD Biosciences), mouse anti-CD31 (Novocastra, Leica Biosystems, Nussloch GmbH, Germany), rabbit anti-VEGFR2 (Abcam, UK), rabbit anti-ionized calcium-binding adaptor molecule-1 (Iba-1) (1:500; FujiFilm Wako Chemicals, VA, USA), mouse anti-glial fibrillary acidic protein (GFAP) (1:500; BD Biosciences), Alexa Fluor 546 donkey anti-rabbit, Alexa Fluor 488 donkey anti-mouse, Alexa Fluor 488 donkey anti-rabbit (all 1:200; Life Technologies) and Alexa Fluor 594 donkey anti-mouse (1:200; Abcam). The nuclei were stained with Hoechst 33342 (4 μg/ml; Molecular Probes, OR, USA). Cell preparations were mounted in Dakocytomation fluorescent medium (Dakocytomation Inc., CA, USA) and images were acquired with AxioImager A1 microscope (Carl Zeiss, Gottingen, Germany).

Briefly, slides were fixed with 4% PFA for 10 min, blocked with 1% BSA/10% normal serum in 0.1% PBS-Tween20 for 30 minutes and immunostained with mouse anti-nestin (Abcam), rabbit anti-VEGFR2 (Abcam) and rabbit anti-GFAP (Abcam) overnight at 4 °C. Alexa 488-conjugated goat anti-mouse and anti-rabbit antibodies (Molecular Probes, Invitrogen, France) were added as secondary reagents. Nuclei were counterstained with DAPI. Samples were subjected to evaluation under a fluorescence microscope.

Slides were incubated with primary antibodies diluted in blocking solution overnight at 4 °C, rinsed, and incubated with the secondary antibodies for 1 h at room temperature. Primary antibodies were the following: rabbit anti-CD31 (1:100, BD Biosciences), rabbit anti- VEGFR2 (1:200, Abcam), rabbit anti-DCX (1:200, Cell Signaling), mouse anti-GFAP (1:200, Abcam), mouse anti-PSA-NCAM (1:200, Millipore), mouse anti-α smooth muscle actin (1:500, Sigma), mouse anti-acetylated α tubulin (1:1000, Sigma), rabbit anti-β catenin (1:500 Abcam), rabbit anti-phosphorylated EGFR (1:200, Cell Signaling), and rabbit anti-Ki67 (1:200, Abcam). For nuclear staining 4′,6-diamidino-2-phenylindole (DAPI) 500 ng/ml (Sigma) was used. Secondary antibodies were Alexa Fluor dye-conjugated goat anti-mouse or goat anti-rabbit IgG or donkey anti-goat IgG (diluted 1:200 in blocking solution, Jackson ImmunoResearch or Invitrogen). Confocal images were taken on LSM 510 META NLO (Carl Zeiss MicroImaging, Inc., Thornwood, NY, USA). The following filter setting is applied: FITC Ch2 band pass (BP) 500–550 IR Texas Red Ch3: long pass (LP) 595 DAPI Ch2: BP 435–485 IR. For low magnification fluorescence micrographs Nikon E800 and 80i upright microscopes with Spot RT cooled CCD cameras were used.