Cells were lysed by treating with CelLytic™ MT Cell Lysis Reagent (Sigma-Aldrich). An NE-PER™ kit (Pierce Biotechnology, Inc., Rockford, IL, USA) was used to isolate nuclear and cytoplasmic proteins. An equimolar quantity of protein was then subjected to SDS-PAGE, following which proteins were transferred onto nitrocellulose filters. The blots were blocked with 2% bovine serum albumin solution for 1 h and subsequently incubated overnight at 4°C with polyclonal goat anti-mouse IL-6 (cat. no. sc-1265; Santa Cruz Biotechnology, Inc.), rabbit anti-mouse IL-6 receptor (IL-6R; cat. no. sc-660; Santa Cruz Biotechnology, Inc.), mouse anti-human VEGF (cat. no. MAB293; R&D Systems, Inc.), rat anti-mouse E-cadherin (cat. no. 748-EC; R&D Systems, Inc.), anti-human mouse STAT3 (cat. no. MAB1799; R&D Systems, Inc.) and polyclonal goat anti-human MMP-9 (cat. no. AF911; R&D Systems, Inc.) (1:1,200 dilution) antibodies. The membranes were further incubated with horseradish peroxidase-conjugated secondary rabbit anti-goat (cat. no. ab6741), rabbit anti-mouse (cat. no. ab6728) or goat anti-rabbit (cat. no. ab6721) IgG antibodies (dilution, 1:1,000–1:2,000; Abcam, Cambridge, MA, USA), for 1 h at room temperature. The film was developed using an X-ray processor and visually examined to determine the intensity of the protein band.
Mouse anti human vegf
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Mouse anti-human VEGF is a primary antibody that specifically recognizes and binds to the human vascular endothelial growth factor (VEGF) protein. VEGF is a key regulator of angiogenesis, the process of new blood vessel formation, and is involved in various physiological and pathological conditions.
Automatically generated - may contain errors
Lab products found in correlation
Cellytic mt cell lysis reagent, by Merck Group (1 mentions)
Rat anti mouse e cadherin, by R&D Systems (1 mentions)
Igg antibody, by Abcam (1 mentions)
Ne per kit, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride filter membrane, by Merck Group (1 mentions)
Odyssey imager, by LI COR (1 mentions)
B iotinylated goat anti mouse immunoglobulin g, by Vector Laboratories (1 mentions)
3 protocols using mouse anti human vegf
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Analysis of Tendon Proteins
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The tendon samples were homogenized. Protein content was normalized and the samples were subjected to SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane filter (Millipore Corp., Billerica, Mass.). The filters were incubated in phosphate-buffered saline containing 0.5% Tween 20 and 5% nonfat milk and then incubated with primary antibody overnight at 4 °C. After incubation with conjugated affinity-purified secondary antibody labeled with IRDye 800, blots were washed and immunoreactive proteins were scanned on an Odyssey imager (LI-COR, Inc., Lincoln, NE). Optical density on the membrane was measured and the relative differences between an internal control (ß-actin) and treated samples were calculated. Mouse anti-rat bFGF (Milipore Corp., Billerica, Mass.), mouse anti-human VEGF (Santa Cruz, Dallas, Texas), mouse anti-chicken MMP2 and TIMP2 (Abcam, Cambridge, Mass.) and mouse anti-chicken type I collagen and type III collagen (Acris, San Diego, Calif.) were used respectively as primary antibodies to detect different proteins.
3
Immunohistochemical Analysis of DLL4 and VEGF
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Colorectal cancer and adenoma tissue was obtained from patients who had undergone surgical treatment or polypectomy. Colorectal adenoma samples were limited to large adenomas (≥10 mm in diameter). Tissue samples were fixed in 10% formalin, embedded in paraffin wax, and cut into 4 µm-thick sections. Immunohistochemical staining was performed using an avidin-biotin-peroxidase (ABC) method. Briefly, after deparaffinizing in xylene and rehydrating in graded ethanol solutions, sections were heated in citrate buffer (0.01 M, pH 6.5) at 120 for 10 min. The antigen retrieval step was identical for each antibody used. Sections were then incubated with mouse anti-human DLL4 (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or mouse anti-human VEGF (1:1000 dilution; Santa Cruz Biotechnology) overnight at 4-8 . Sections were washed four times with 0.1 M phosphate buffered saline (PBS; pH 7.4), then incubated with biotinylated goat anti-mouse immunoglobulin G (1:200 dilution; Vector Laboratories, Burlingame, CA, USA) at room temperature for 90 min. Slides were washed three times with 0.1 M PBS (pH 7.4) and immunoreactive staining was visualized using 0.5 mg/ml 3,3′-diaminobenzidine tetrahydrochloride and 0.03% hydrogen peroxide. Bovine serum albumin was used in place of primary antibody, as a negative control.
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