The paraffin-embedded tumor sections were stained for anti-Ki67, anti-PCNA, anti-MMP-9, and anti-VEGF (Abcam). Sections (2 µm) were deparaffinized and pretreated with citrate buffer using a heat-induced epitope retrieval protocol. Endogenous peroxidase was blocked with 20% hydrogen peroxide for 15 minutes at room temperature followed by incubation with anti-Ki67, anti-PCNA, anti-MMP-9, and anti-VEGF for 30 minutes, respectively. A biotinylated goat anti-mouse immunglobulin G secondary antibody (Dako, Glostrup, Denmark) was then applied to each slide for 30 minutes. After washing in Tris-hydrochloric acid buffer, the slides were incubated with peroxidase-conjugated streptavidin complex reagent (Dako) and developed with 3,3′-diaminobenzidine for 5 minutes. The slides were counterstained and dehydrated.
Anti vegf
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Anti-VEGF is a protein that binds to and neutralizes the activity of vascular endothelial growth factor (VEGF), a key regulator of angiogenesis. It is commonly used in research applications to study the role of VEGF in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Abcam (13 mentions)
Pvdf membrane, by Merck Group (11 mentions)
Anti mmp9, by Abcam (9 mentions)
Anti hif 1α, by Abcam (8 mentions)
Anti gapdh, by Abcam (8 mentions)
Anti cd31, by Abcam (8 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Anti ki67, by Abcam (6 mentions)
Anti cd9, by System Biosciences (6 mentions)
Anti cd63, by System Biosciences (6 mentions)
Anti cd81, by System Biosciences (6 mentions)
Anti p akt, by Abcam (5 mentions)
Anti akt, by Abcam (5 mentions)
β actin, by Abcam (5 mentions)
Anti β actin, by Merck Group (5 mentions)
Anti bcl 2, by Cell Signaling Technology (4 mentions)
Anti vegfr2, by Cell Signaling Technology (4 mentions)
Anti cd34, by Abcam (4 mentions)
Nitrocellulose membrane, by Bio-Rad (4 mentions)
Anti e cadherin, by Abcam (4 mentions)
119 protocols using anti vegf
1
Immunohistochemical Analysis of Tumor Markers
2
Comprehensive IHC Assay Protocol
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IHC assays were performed as previously described [13 (link)]. For primary antibody incubation, anti-PTBP3 (Santa Cruz Biotechnology) antibody was applied at 1:100 dilution, anti-HIF-1α (MA1–16511, Thermo Fisher) antibody at 1:100 dilution, anti-CD31 (11265–1-AP, Proteintech, China) antibody at 1:50 dilution, anti-VEGF (Abcam) antibody at 1:100 dilution.
3
Quantifying Osteogenic and Angiogenic Factors
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BMP-2, Col-1, OCN, VEGF, and bFGF protein levels in the PDCs lysates were determined by Western blot analysis. The cells were homogenized using SDS lysis buffer for 30 min on ice. The protein concentration was measured by BCA assay. Then, 50 μg of total protein per sample were run on SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked with Tris-buffered saline containing 5% non-fat milk at room temperature for 2 h and incubated with the following primary antibodies at 4 °C overnight: anti-BMP-2, anti-Col-1, anti-OCN, anti-VEGF (all from Abcam, USA; 1:1000), and anti-bFGF (Biosynthesis Biotechnology, China; 1:350). After washing thrice in TBST buffer, the membranes were incubated with HRP-conjugated secondary antibody (Boster Biotechnology, China; 1:1500) for 2 h at room temperature. The chemical luminescence reaction was measured by ECL. To confirm equal protein loading, the same blots were incubated with antibodies specific for GAPDH/β-actin (Santa Cruz, USA; 1:1000). The target bands on the membranes were analyzed by a gel imaging system with the reference OD values. The experiments were performed independently and repeated three times and the relative OD values were calculated.
4
Immunohistochemical Evaluation of Corneal Markers
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IHC for COX-2, VEGF, and MMP-9 was performed according to the protocol described previously [40 (link)] from the lab. Briefly, the corneal sections were first rehydrated, then heat-mediated epitope retrieval was performed, endogenous peroxidase activity was blocked, and sections were incubated with the respective primary antibodies [anti-VEGF (Cat#ab28775), anti-MMP-9 (Cat#ab58803) both from Abcam, Cambridge, MA, and anti-COX-2 (cat#160112; Cayman chemicals, Ann Arbor, MI); Rabbit IgG antibody (N-Universal, DAKO), negative control]. Thereafter, the sections were incubated sequentially using the streptavidin-biotin complex (ABC) IHC staining methodology for signal amplification. DAB was used for visualization of IHC (brown, cytoplasmic staining) followed by hematoxylin staining for the nucleus. The scoring for the cytoplasmic staining was performed in 10 randomly selected areas (400x magnification). This intensity of IHC staining was scored from 0 to 4; where a score of 0 signified no DAB staining (absence of any brown color) and 4 signified the maximum intensity of staining, described previously [40 (link)].
5
Protein Extraction and Western Blot Analysis
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For protein extraction, cells were lysed in cell lysis buffer containing 140 mM NaCl, 10 mM Tris-HCl, 1% Triton X-100, 1 mM ECTA and 1X protease inhibitor cocktail (Gibco; Thermo Fisher Scientific, Inc.). The protein concentration was determined using the BCA assay. A total of 20 µg protein was loaded per lane and separated on 12% SDS-PAGE gels. Proteins were subsequently transferred to polyvinylidene difluoride membranes, which were blocked with 5% non-fat dry milk for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: Anti-GAPDH (cat no. sc-25778; 1:2,000) anti-BMP6 (cat no. sc-7406; 1:100; both Santa Cruz Biotechnology, Inc.) and anti-VEGF (cat no. ab105219; 1:1,000; Abcam). They were subsequently incubated with an anti-goat horseradish peroxidase (HRP)-conjugated secondary antibody (cat no. sc-2004, 1:1,000, Santa Cruz Biotechnology, Inc.) at 4°C for overnight, and detected with an enhanced chemiluminescence detection kit (EMD Millipore, Billerica, MA, USA).
6
DMOG Modulates HIF-1α and VEGF in hiPSC-MSCs
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hiPSC-MSCs cultured with or without 1000 μM DMOG under normal oxygen conditions were analyzed by western blotting. The total cellular proteins were first extracted. Briefly, the cells were lysed in ice-cold lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 0.1% SDS) containing 1 mM phenylmethylsulphonyl fluoride (PMSF) (Sigma, USA) and phosphatase inhibitor cocktail (Sigma, USA). The cell lysates were cleared by centrifugation at 4°C and 12,000 rpm for 15 min. The protein concentrations of the lysates were quantified using a BCA assay Kit. The cell proteins were separated by standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. After incubation in 5% BSA blocking solution for 1 h, the membranes were incubated overnight at 4°C with anti-HIF-1α, anti-VEGF, anti-p-Akt, anti-Akt, and anti-β-actin antibodies (all from Abcam, UK). The signal inhibitor used for PI3K/Akt was LY294002 (20 μM; Sigma, USA). The membranes were then washed three times with PBS-Tween-20 and incubated with horseradish peroxidase (HPR)-conjugated secondary antibodies (Sigma, USA) at 37°C. The immunoreactive bands were visualized using the ECL chemiluminescence reagent (Millipore, USA).
7
Comprehensive Protein Expression Analysis
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The primary antibodies used were anti-collagen I, anti-VEGF and anti-PDGFBB (Abcam, Cambridge, UK); anti-TSG101, anti-CD9, anti-CD63, and anti-CD81 (System Biosciences, Mountain View, CA, USA). The primary antibodies anti-CD41, anti-TGF-β, anti-bFGF, anti-cleaved-caspase-3, anti-Runx2, anti-β-catenin, anti-β-tubulin, anti-calnexin, anti-Bcl-2, anti-CHOP, anti-Bad and phosphorylated Bad (p-Bad), anti-Erk1/2 and phosphorylated Erk1/2 (p-Erk1/2), anti-Akt and phosphorylated Akt (p-Akt) antibody were all obtained from Cell Signaling Technology (Danvers, MA, USA). The anti-PERK and phosphorylated PERK (p-PERK) antibodies were obtained from Santa Cruz Biotechnology.
8
Western Blot Analysis of Angiogenic Markers
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Western blot analyses were performed as previously described (X. P. Chen et al., 2016 ) with the following primary antibodies: anti‐endocan (0.1 μg/ml; R&D Systems); anti‐ERK1/2 (1:1,000; Cell Signaling Technology); anti‐p‐ERK1/2 (1:1,000; Cell Signaling Technology); anti‐VEGF (1:1,000; Abcam); anti‐VEGFR1 (1:1,000; Abcam); anti‐VEGFR2 (1:1,000; Cell Signaling Technology); and GAPDH (1:1,000; Cell Signaling Technology).
9
Immunohistochemical Analysis of Callus Vascularization
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To assess blood vessel formation of the callus, vascular endothelial growth factor (VEGF) immunohistochemistry was performed 4 weeks after injection. Additional slices were firstly deparaffinized, dehydrated, rinsed and incubated with 3% hydrogen peroxide for 20 min. Trypsin-induced epitope retrieval was performed for 20 min at room temperature. All sections were then blocked with 0.1% BSA in PBS for 1 h. Thereafter, the callus sections described above were incubated with primary antibodies (anti-VEGF; Abcam, Cambridge, UK) overnight at 4°C and then incubated with horseradish peroxidase-coupled secondary antibodies (Aspen, Guangzhou, People’s Republic of China).
10
Quantifying Osteogenic and Angiogenic Factors
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Osteogenic proteins (RUNX2 and ALP) and angiogenic proteins (VEGF and TGF-β1) were measured using Western blot analysis in 4 and 6 weeks postoperatively. Western blot was performed according to the manufacturer’s protocol. The primary antibodies including anti-RUNX2 (1:1000), anti-ALP (1 μg/ml), anti-VEGF (5 μg/ml), anti-TGF-β1 (1:300), and β-actin (1:5000) were obtained from Abcam (Cambridge, UK). The immunoreactive bands were visualized and finally quantified using Quantity One Software. And all values were normalized to the value of β-actin.
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