Cells were grown to 80-90% confluency in 15-cm dishes coated with 10 μg/ml FN in PBS. After starvation in OptiMEM® for 3 h, cells were stimulated with 30 ng/ml VEGF for 10 min (+VEGF), or for the indicated times, at 37°C. Cells were then placed on ice, washed two times with PBS, and lysed in 0.5 ml/plate of RIPA buffer (20 mM Tris pH 7.4, 50 mM NaCl, 0.1% SDS, 1% Triton, 1% Deoxycholate, 1% NP40) containing PMSF (∼1 mM) and Halt® Protease and Phosphatase inhibitor (1:100). Lysates were centrifuged at 12,000 g for 10 min at 4°C. 400 μg of total protein from each sample was immunoprecipitated by incubating them with protein-G Dynabeads® (Invitrogen) coupled to a rabbit-anti-mouse-VEGFR2 antibody (clone 55B11, Cell Signaling Technology) for the VEGFR2 immunoprecipitation (IP), or a goat anti-mouse Neuropilin-1 antibody (AF566, R&D Systems) for the NRP1 IP, on a rotator overnight at 4°C. Immunoprecipitated complexes were washed three times with 0.2 ml of RIPA buffer, and once in PBS, before being added to, and boiled in, 1× NuPAGE® sample reducing agent (Life Technologies), ready for western blotting or mass spectrometry analysis.
Rabbit anti mouse vegfr2 antibody
Manufactured by Cell Signaling Technology
The Rabbit-anti-mouse-VEGFR2 antibody is a research-use laboratory reagent produced by Cell Signaling Technology. It is designed to detect the mouse vascular endothelial growth factor receptor 2 (VEGFR2) protein in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Rat anti mouse cd31 antibody, by Thermo Fisher Scientific (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Axiocam mrc, by Zeiss (2 mentions)
Protein g dynabead, by Thermo Fisher Scientific (1 mentions)
Nupage sample reducing agent, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline solution, by Merck Group (1 mentions)
Cm1950 cryostat microtome, by Leica (1 mentions)
Fitc conjugated anti rat secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Tcs sp5, by Leica (1 mentions)
3 protocols using rabbit anti mouse vegfr2 antibody
1
VEGFR2 and NRP1 Immunoprecipitation
2
Quantifying Tumor Vasculature and VEGFR2
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At day 10, mice were sacrificed for tumor ex-vivo analysis. After 24-h fixation in a solution of 4 % paraformaldehyde and phosphate-buffered saline, followed by 3-day fixation in 30 % sucrose and phosphate-buffered saline solution (Sigma-Aldrich, St Louis, MO), tumors were sectioned into 10-mm slices for immunofluorescence staining. Rabbit antimouse VEGFR2 antibody (Cell Signaling, Danvers, MA) and rat antimouse CD31 antibody (eBioscience, San Jose, CA) were used to quantity VEGFR2 expression and the percentage blood vessel volume, respectively. Fluorescent microscopy was performed with an LSM510 meta-confocal microscope (Zeiss, Maple Grove, MN) attached to a digital camera (AxioCam MRc, Bernried, Germany) using a × 20 objective. On each histological slice, five fields of view (FOVs) of 0.19 mm2 were randomly selected and the VEGFR2 expression and the percentage blood vessel area per FOV were quantified with ImageJ software (National Institutes of Health, Bethesda, MD) as the average value in the five FOVs.
3
Dual Immunofluorescence for VEGFR2 and CD31
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After ultrasound imaging, the mice were sacrificed immediately and uterus were harvested for the histological study. Double staining for VEGFR2 and CD31 was performed to confirm co-localization of VEGFR2 on CD31-positive vascular endothelial cells. Briefly, the dissected uterus samples were covered with Tissue-Tek (Sakura), and then frozen in liquid nitrogen vapor. The tissue sections (10 μm) were cut with a cryostat microtome (CM1950; Leica, Heidelberg, Germany) and fixed with pre-cooled acetone for 2 min, followed by drying in air for at least 1 h. Then, the sections were rinsed with PBS for 5 min and incubated with 0.03% H2O2 in PBS, and subsequently blocked with 5% goat serum for 1 h at room temperature. After that, slides were co-incubated with rabbit anti-mouse VEGFR2 antibody (Cell Signaling Technology Inc., Danvers, MA) and rat anti-mouse CD31 antibody (eBioscience, San Diego, CA) at a dilution of 1:200 overnight at 4°C. Cy3-conjugated anti-rabbit (biorbyt, Cambridge, UK) and FITC-conjugated anti-rat secondary antibodies (eBioscience, San Diego, CA) were used to visualize the expressions of VEGFR2 and CD31, respectively. Fluorescent images were acquired at × 200 magnifications with a laser scanning confocal microscope (TCS SP5, Leica, Germany).
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