Tumor samples were weighted and homogenized in buffer TBS 0.1% BSA with the addition of a protease inhibitor cocktail (Sigma, Saint Louis, Missouri) in a volume proportional to their weight. Cell supernatants were assayed after centrifugation at 2,000 x g RT for 5 min. ELISA with anti-mouse-VEGF (R&D Systems, Minneapolis, Minnesota) was performed following the manufacturer’s instructions. VEGF levels from tumor samples were normalized based on lysate protein concentration.
Anti mouse vegf
Manufactured by R&D Systems
Sourced in United States
Anti-mouse-VEGF is a lab reagent used to detect and measure mouse Vascular Endothelial Growth Factor (VEGF) levels in various sample types. It functions as a capture antibody in immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (1 mentions)
Anti mouse il 10, by R&D Systems (1 mentions)
Normal goat igg control, by R&D Systems (1 mentions)
Anti human vegf, by R&D Systems (1 mentions)
Mouse anti albumin, by Santa Cruz Biotechnology (1 mentions)
Clarity western ecl kit, by Bio-Rad (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Rabbit anti β actin, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Rabbit anti crp, by Santa Cruz Biotechnology (1 mentions)
Streptavidin horseradish peroxidase hrp, by Thermo Fisher Scientific (1 mentions)
Goat anti vegf, by R&D Systems (1 mentions)
4 protocols using anti mouse vegf
1
Quantifying Tumor VEGF Levels
2
Blocking Antibodies and Mcl-1 Inhibitor Treatment
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The following blocking antibodies were used: 10–20 μg/mL anti-mouse GM-CSF blocking antibody (eBioscience, San Diego, CA, USA), 20 μg/mL anti-mouse VEGF, 20 μg/mL anti-mouse IL-10, 10 μg/mL anti-human GM-CSF, 10 μg/mL anti-human VEGF and 20 μg/mL anti-human IL-10 blocking antibodies (R&D systems, Minneapolis, MN, USA). Isotype controls used were 10 μg/mL rat IgG2aK isotype control (eBioscience) and normal goat IgG control (R&D). Mcl-1 inhibitor MIM1 (R&D) was dissolved in DMSO.
3
Western Blot Analysis of Liver Proteins
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Cells and liver tissues were lysed in RIPA buffer containing protease inhibitor cocktail (Roche) and phosphatase inhibitor (Sigma-Aldrich). A total of 45 ug protein extracts were separated in 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE). The separated proteins were transferred onto PVDF membranes (Bio-Rad, Hercules, CA, USA). The membrane was incubated with rabbit anti-CRP (1:1,000, Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-albumin (1:1,000 Santa Cruz), mouse anti-VEGF, rabbit anti-VEGFR1, mouse anti-endoglin (1:1,000 R&D Systems, Abingdon, UK), and rabbit anti-β-actin (1:3,000, Sigma Aldrich) at 4℃ overnight. The membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (anti-rabbit IgG [1:20,000, Bio-Rad Laboratories, Hercules, CA, USA] or antimouse IgG [1:5,000, Santa Cruz]) for 1 hour at room temperature. The bands were detected using Clarity Western ECL kit (Bio-Rad).
4
Quantifying VEGF Secretion via ELISA
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ELISA high binding plates (Thermo Fisher Scientific) were coated with 1 μg/mL of mouse anti‐VEGF (R&D Biosystems) in phosphate‐buffered saline (PBS) overnight at room temperature. Plates were then blocked with 300 μL of blocking buffer (1% BSA in PBS) for 1 hour at room temperature. After washing with wash buffer (PBS with 0.2% Tween‐20), 100 μL samples of conditioned medium from siRNA‐transfected 60 mm plates were added to appropriate wells and incubated at room temperature for 2 hours. A standard curve of recombinant VEGF from 0 to 2000 pg/mL was run alongside experimental samples. Plates were washed again and incubated with 1.2 μg/mL goat anti‐VEGF (R&D) for 2 hours at room temperature. After washing, streptavidin‐horseradish peroxidase (HRP, Thermo Fisher Scientific) was added for 1 hour followed by detection with 3,3′,5,5′‐tetramethylbenzidine (TMB, eBioscience). Data were analysed using one‐way ANOVA with Tukey's post hoc testing.
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