Immediately after 2-h ischemia, rat was perfused with ice-cold PBS followed by 4% PFA. Analysis of VEGF was carried out by immunostaining using the 20-μm-thick cryosection as described (Wang et al., 2017 (link)). In brief, tissue was pre-incubated for 1 h at room temperature in PBS containing 0.1% Triton X-100, 1% BSA, and 5% goat serum to block non-specific binding sites. Then anti-VEGF antibody (1:200, Abcam), anti-NeuN (1:200, Millipore), anti-GFAP (1:2,000, Millipore) primary antibody were applied to the sections and incubated at 4°C overnight. Cy3 conjugated secondary antibody (anti-mouse, 1:800) or 488-conjugated secondary antibody (anti-rabbit, 1:800) was incubated with the brain cryosection for 2 h at room temperature. The staining was visualized under LSM 700 confocal laser-scanning microscope (Zeiss), and images were taken from the ischemic and the mirrored non-ischemic region.
Anti vegf antibody
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-VEGF antibody is a laboratory reagent used to detect and quantify the presence of vascular endothelial growth factor (VEGF) in biological samples. It functions by binding specifically to VEGF proteins, enabling their identification and measurement through various analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Anti gfap, by Merck Group (2 mentions)
Anti neun, by Merck Group (2 mentions)
Lsm 700 confocal laser scanning microscope, by Zeiss (2 mentions)
Anti hif 1α antibody, by Abcam (2 mentions)
Anti β actin antibody, by Abcam (2 mentions)
Horseradish peroxidase conjugated anti mouse antibody, by Santa Cruz Biotechnology (2 mentions)
Diaminobenzidine tetrahydrochloride, by Santa Cruz Biotechnology (2 mentions)
Primary anti collagen type 1 antibody, by Abcam (2 mentions)
Anti collagen type x antibody, by Abcam (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Anti ki67 antibody, by Abcam (2 mentions)
Anti cd31 antibody, by Abcam (2 mentions)
Anti p300 antibody, by Santa Cruz Biotechnology (1 mentions)
Anti histone h3 antibody, by Merck Group (1 mentions)
Anti creb antibody, by Santa Cruz Biotechnology (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Blocking buffer, by Agilent Technologies (1 mentions)
Anti runx2 antibody, by Abcam (1 mentions)
Anti collagen type 2 antibody, by OriGene (1 mentions)
33 protocols using anti vegf antibody
1
VEGF Immunostaining in Rat Ischemic Brain
2
Western Blot Analysis of Protein Markers
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Protein samples were separated in 8% (or 12%) sodium dodecyl sulfate polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene difluoride membranes. The primary antibodies used are: anti‐HIF‐1α antibody (1:1,000; Abcam, Cambridge, Massachusetts, USA), anti‐β‐actin antibody (1:25,000; Abcam), anti‐VEGF antibody (1:1,000; Abcam), anti‐p300 antibody (1:1,000; Santa Cruz, Santa Cruz, California, USA), anti‐CREB antibody (1:500; Santa Cruz) and anti‐Histone H3 antibody (1:2,000; Millipore, Billerica, Massachusetts, USA).
3
Immunofluorescence Analysis of Hypoxia-Induced Markers
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Cells (5 × 104 cells/well) were seeded into a 24-well plate and cultured under normoxic or hypoxic conditions for 24 h. After 4% paraformaldehyde (PFA) (Sigma-Aldrich) fixation for 30 min, cells were incubated with blocking buffer (Dako, Denmark) at 37°C for 1 h, followed by incubation with anti-HIF-1α antibody (1:300), anti-VEGF antibody (1:300), or anti-RUNX2 antibody (1:300) (all from Abcam) at 4°C overnight. After washing with phosphate-buffered saline (PBS), cells were incubated with goat anti-rabbit or mouse IgG antibody conjugated with Alexa 488 or Alexa 588 (1:500) (Invitrogen) for 1 h at room temperature. Cells were then stained with 4,6-diamidino-2-phenylindole (DAPI) and observed under a fluorescence microscope (Olympus, Japan). For tissue immunofluorescence, rat periodontal tissues of the right maxillary first molar were excised, frozen, sectioned, and antigen retrieved, which was followed by the steps listed above.
4
Histological Analysis of Cartilage Tissue
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Samples were fixed in 4% paraformaldehyde and embedded in paraffin and then cut into 8 um sections. Sections were stained with hematoxylin and eosin (H & E), safranin-O, Masson trichrome or Von Kossa. To investigate the expression of type I, type II and type X collagen in matrices and vascular endothelial growth factor (VEGF), some sections were processed for two-step indirect immunohistochemical staining as studied previously [17 (link)]. Briefly, expression of type I collagen, type II collagen, type X collagen and VEGF was detected using primary anti-collagen type I antibody (mouse anti-rabbit, 1:50; Abcam, Cambridge, MA USA), anti-collagen type II antibody (mouse anti-rabbit, 1:50; Acris, Herford Germany), anti-collagen type X antibody (mouse anti-rabbit, 1:50; Abcam) and anti-VEGF antibody (mouse anti-rabbit, 1:50; Abcam), followed by horseradish peroxidase-conjugated anti-mouse antibody (1:200 in PBS; Santa Cruz Dallas, Texas, USA) and color development with diaminobenzidine tetrahydrochloride (Santa Cruz).
5
Protein Expression Analysis in Cell Cultures
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Total proteins were extracted using the Total Protein Extraction kit (KeyGen Biotech) on days 3 and 6. Lysates were precipitated at 12,000 rpm for 15 min and the total protein content was assessed using the BCA Protein Assay kit (KeyGen Biotech). Proteins were separated by electrophoresis through SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane, and the membranes were then blocked and incubated with mouse monoclonal antibody [ACTN05 (C4)] to actin (Cat. no. ab3280), rabbit monoclonal antibody [EP708Y] to C/EBPα (Cat. no. ab40761) (both from Abcam), anti-peroxisome proliferator sctivated receptor γ, isoform 1and 2 antibody (Cat. no. MAB3872; Millipore Corp., Billerica, MA, USA), rabbit polyclonal antibody to adiponectin (Cat. no. ab62551; Abcam), fatty acid binding protein 4 (FABP4) antibody (Cat. no. 220367; Zen-Bio, Inc., Durham, NC, USA) and anti-VEGF antibody (Cat. no. ab46154; Abcam) at 4°C overnight. The results were visualized with Immobilon Western Chemiluminescent HRP Substrate (Millipore Corp.) after they were incubated with the secondary antibody. The secondary antibodies were peroxidase-conjugated goat anti-rabbit lgG (H+L) (Cat. no. ZB-2301) and peroxidase-conjugated goat anti-mouse lgG (H+L) (Cat. no. ZB-2305) (both from ZSGB-BIO, Beijing, China).
6
Western Blot Analysis of Osteogenic Markers
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The cells were treated with the lysis buffer (Cell Signaling Technology), and the protein extracts were dissolved in a sample buffer containing 50 mM Tris-HCl, 2% SDS, 10% glycerol, and 100 mM dithiothreitol (pH = 6.80). Proteins were separated using SDS-PAGE in 10% polyacrylamide gel and transferred to a nitrocellulose membrane. Blots were performed with anti-BMP-2 antibody, anti-VEGF antibody, anti-ALP antibody (Abcam), anti-β-catenin antibodies, anti-GSK-3β antibodies, and anti-C-myc antibodies (Santa Cruz Biotechnology, CA). The bands were captured and documented using a CCD system (Image Station 2000 MM, Kodak, Rochester, NY, USA). The blots were stripped and reprobed with anti-actin antibodies to demonstrate equal loading and to enable between-group protein content normalization. Densitometry of the bands was performed using Molecular Imaging Software Version 4.0 (Kodak).
7
Cranial Base Angiogenesis Regulation
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Skulls of 15 day-old mice were fixed and paraffin embedded. Fresh 7 μm sagittal setions of the cranial base were immunostained using Annexin V antibody (Abcam), anti VEGF antibody (Abcam), or cleaved caspase 3 antibody (Cell Signaling). After primary antibody incubation, sections were incubated with Alexafluor 555 goat anti-rabbit IgG (Molecular Probes), then mounted in ProLong Gold antifade reagent with DAPI (Invitrogen). Images were captured using Nikon Eclipse E800 microscope. Image J software was used for immunofluorescent stain quantification to calculate means and standard deviations per genotype (n = 3 mice per genotype).
8
Histological Analysis of Calcium Deposition and VEGF/BMP-6 Expression
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Slides were washed in xylene twice for 10 min each in order to remove the paraffin. A graded series of ethanol solutions, including 100, 95, and 70% ethanol were used for rehydration. The sections were washed twice in deionized water for 5 min each. Tissues were fixed in 4% paraformaldehyde at 4°C for overnight, embedded in paraffin, sliced into sections 4 mm thick and stained with hematoxylin and eosin (H&E) at room temperature for 15 min. For Von Kossa staining, sections were stained with von Kossa stain at room temperature for 20 min to examine calcium deposition. In addition, immunohistochemistry was used to examine the expression of VEGF and BMP-6 in implants. Fixed tissues were deparaffinized, incubated in citrate buffer at room temperature for 10 min and subsequently incubated with the primary antibodies, including anti-BMP6 antibody (cat no. ab155963; 1:200; Abcam) and anti-VEGF antibody (cat no. ab81289; 1:200; Abcam) overnight at 4°C, followed by biotinylated goat anti-mouse immunoglobulin G secondary antibodies (cat no. BA1300; 1:500, BD Biosciences) and treated with streptavidin-horseradish peroxidase (BD Biosciences). Tissues were then treated with 3,3′-diaminobenzidine substrate and hematoxylin for 15 min at room temperature. The data was detected using ImageJ software 4.8 (National Institutes of Health, Bethesda, MA, USA).
9
Antibody and Reagent Purchase Protocol
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The Anti-VEGF antibody was purchased from abcam. DNAs and siRNAs were purchased from Sangon Biotech (Shanghai) and Jinweizhi Company. DOX were purchased from Aladdin Company (Shanghai, China).
10
Quantifying TRPC4, NF-κB, and VEGF Levels
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After 48 h of transfection with TRPC4-siRNA or NC-siRNA, total protein was extracted from the HCAECs, and the quantity of protein was determined using the BCA assay kit (Beyotime, Shanghai, China). The protein samples were then separated on 10–12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were then blocked with 5% non-fat milk for 2 h at room temperature and incubated overnight at 4°C with the following antibodies: anti-TRPC4 antibody (Millipore, Bedford, MA, USA, #AB15302), anti-nuclear factor (NF)-κB p65 antibody (Santa Cruz, SC-7151), or anti-VEGF antibody (Abcam, Cambridge, MA, USA, ab46154). The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Abcam, ab97023). GAPDH was used as loading control. Immunoreactive proteins were visualized using an enhanced chemiluminescence (ECL) chemiluminescent detection system (Pierce ECL Western Blotting Substrate detection system; Thermo Scientific).
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