For the immunohistochemical staining, the tumors were resected and fixed in a 10% formalin solution 4 days after the hyperthermia treatment. The tumor tissues were sectioned to a 5-μm thickness and fixed to slides. The slides were deparaffinized and incubated with 10% normal serum for 30 min to block background staining. The slides were then incubated for 60 min at 37°C with a rabbit anti-VEGF polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) or a rabbit anti-cluster of differentiation (CD)34 polyclonal antibody (Boster Biological Technology, Ltd., Wuhan, China), and subsequently incubated with horseradish peroxidase-conjugated secondary antibodies. Each step was followed by washing with phosphate-buffered saline three times. Peroxidase activity was visualized by treatment with 0.02% diaminobenzidine tetrahydrochloride solution containing 0.005% hydrogen peroxide at room temperature for 5–10 min. The sections were also counterstained with hematoxylin and eosin. Iron in the magnetic nanoparticles was stained with prussian blue. Protein quantification was performed by Imagepro-plus 6.0 software (Media Cybernetics, Rockville, MD, USA), and the intensity values were normalized to the background.
Rabbit polyclonal anti vegf antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Rabbit polyclonal anti-VEGF antibody is a laboratory reagent designed to detect and measure the Vascular Endothelial Growth Factor (VEGF) protein in biological samples. It is a polyclonal antibody produced in rabbits that specifically binds to the VEGF protein.
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Lab products found in correlation
Rabbit polyclonal anti vwf antibody, by Abcam (2 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Biotinylated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
3 3 diaminobenzidine (dab), by Maixin Group (1 mentions)
Rabbit anti ccn1 polyclonal antibody, by Abcam (1 mentions)
B201 optical microscope, by Olympus (1 mentions)
Kaiser s glycerol gelatin, by Merck Group (1 mentions)
Dako antibody diluent, by Agilent Technologies (1 mentions)
Anti nf κb p65, by Santa Cruz Biotechnology (1 mentions)
Anti 8 ohdg antibody, by Abcam (1 mentions)
Polyoxyethylene sorbitan monolaurate, by Nacalai Tesque (1 mentions)
Axiovert 200, by Zeiss (1 mentions)
Procarta immunoassay kit, by Thermo Fisher Scientific (1 mentions)
Avidin biotin complex kit, by Vector Laboratories (1 mentions)
Diaminobenzidine substrate kit, by Vector Laboratories (1 mentions)
Bio plex multiplex assay system, by Bio-Rad (1 mentions)
Rabbit monoclonal anti gapdh antibody, by Cell Signaling Technology (1 mentions)
Anti cd31 rabbit polyclonal antibody, by Abcam (1 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
8 protocols using rabbit polyclonal anti vegf antibody
1
Immunohistochemical Analysis of VEGF and CD34
2
Immunohistochemical Analysis of Eye Tissue
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Formalin-fixed, paraffin-embedded 6 μm eye tissue sections were placed on slides, deparaffinized in xylene, and rehydrated in graded ethanol baths in phosphate-buffered saline. Immunostaining was performed by the streptavidin-peroxidase method (Ultrasensitive; MaiXin, Fuzhou, People’s Republic of China). Hydrogen peroxide (3%) was applied to block endogenous peroxidase activity, and normal goat serum was used to reduce nonspecific binding.
Sections were incubated with commercially available primary antibodies: rabbit anti-CCN1 polyclonal antibody (1:500, Abcam, Cambridge, UK); rabbit anti-p-AKT1/2/3 (Ser473) polyclonal antibody (1:500, Santa Cruz Biotechnology Inc., Dallas, TX, USA); or rabbit anti-VEGF polyclonal antibody (1:500, Santa Cruz Biotechnology Inc.) overnight at 4°C. The sections were incubated with biotinylated secondary antibody (1:1,000, Santa Cruz Biotechnology Inc.) and reacted with the avidin-biotinylated peroxidase complex. The primary antibody was replaced with phosphate-buffered saline for negative controls. The peroxidase reaction was developed with 3, 3′-diaminobenzidine (Maixin Biotechnology, Foshan, China), and sections were counterstained with hematoxylin, dehydrated with alcohol, and mounted using a standard procedure. Images were digitally captured using an Olympus B201 optical microscope.
Sections were incubated with commercially available primary antibodies: rabbit anti-CCN1 polyclonal antibody (1:500, Abcam, Cambridge, UK); rabbit anti-p-AKT1/2/3 (Ser473) polyclonal antibody (1:500, Santa Cruz Biotechnology Inc., Dallas, TX, USA); or rabbit anti-VEGF polyclonal antibody (1:500, Santa Cruz Biotechnology Inc.) overnight at 4°C. The sections were incubated with biotinylated secondary antibody (1:1,000, Santa Cruz Biotechnology Inc.) and reacted with the avidin-biotinylated peroxidase complex. The primary antibody was replaced with phosphate-buffered saline for negative controls. The peroxidase reaction was developed with 3, 3′-diaminobenzidine (Maixin Biotechnology, Foshan, China), and sections were counterstained with hematoxylin, dehydrated with alcohol, and mounted using a standard procedure. Images were digitally captured using an Olympus B201 optical microscope.
3
Quantification of VEGF Expression
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VEGF expression was conducted with an anti-VEGF rabbit polyclonal antibody (Santa Cruz Biotechnology) at a 1:50 dilution and reported as a percentage (%) of positive cells with the respect to a total of at least 1000 tumor cells [40 (link)].
4
Immunohistochemical Analysis of VEGF Expression
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The Liver samples were embedded and frozen in a Tissue-Tek (Sakura, Zoeterwoude, Netherlands) and 5 μM cryo-sections were prepared. These Sections were fixed in 100 % acetone and equilibrated in PBS followed by overnight incubation at 4 °C with anti-VEGF rabbit polyclonal antibody (Santa Cruz Biotechnology Inc., USA) which was diluted with Dako antibody diluents (DAKO, Hamburg, Germany). The cryosections were then incubated with Anti-rabbit Alkaline Phosphatase supervision polymer system (DCS Innovative Diagnostik-Systeme, Hamburg, Germany). Staining was visualized using the Neu Fuchsin substrate Chromogen (DCS Innovative Diagnostik-Systeme, Hamburg, Germany) and were counterstained with Hematoxylin and mounted in Kaisers Glycerol Gelatin (Merck, Darmstadt, Germany). To evaluate the expression of VEGF, all slides were examined and scored by two independent pathologists who were blinded to the animal data. The percentage staining was scored as follows: 0 (No staining, 1 (0-5 %), 2 (6-25 %), 3 (26-50 %), 4 (51-75 %), 5 (76-100 %).
5
Immunohistochemical Analysis of Oxidative Stress
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Immunohistochemical staining was performed on 5-μm-thick paraffin sections using immunoperoxidase visualization. After routine deparaffinization, heat-induced epitope retrieval was performed by immersing the slides in 0.01 M sodium citrate buffer (pH 6.0). The sections were then incubated for 20 h at 4°C with mouse monoclonal anti-8-OHdG antibody (Abcam, Cambridge, MA, USA), anti-NF-κB-p65 (Santa Cruz Biotechnology), rabbit polyclonal anti-vWF antibody (Abcam), or rabbit polyclonal anti-VEGF antibody (Santa Cruz Biotechnology) as primary antibodies. Sections were the washed in PBS for 10 min at three times and then incubated with the suitable fluorochrome-labeled secondary antibody after being probed with the primary antibody. Each selected frame was a representative image obtained from serial confocal Z-sections (where the Z-interval between two sections was 0.2 µm) within a stack of 20 sections.
6
Immunofluorescence Analysis of VEGF and Apelin
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Following fixation, SM samples were embedded in paraffin, sectioned at 3 μm thickness, then deparaffinized (Clear Plus®, FALMA, Tokyo, Japan) and pretreated with sodium citrate buffer (pH 6.0) containing 0.1% polyoxyethylene sorbitan monolaurate (Nacalai Tesque, Kyoto, Japan) at 98 °C for 20 min for antigen retrieval. The sections were subsequently washed three times with phosphate-buffered saline for 5 min and incubated with rabbit polyclonal anti-VEGF antibody (1:100 dilution; Santa Cruz Biotechnology Inc., Santa Cruz CA, USA) and mouse monoclonal anti-apelin antibody (1:100 dilution; Santa Cruz Biotechnology) for 4 h at 4 °C. The sections were additionally incubated with Alexa 488 Fluor®-conjugated goat anti-rabbit IgG antibody (1:100 dilution; Thermo Fisher Scientific, Waltham MA, USA) and Alexa 594 Fluor®-conjugated goat anti-mouse IgG antibody (1:100 dilution; Thermo Fisher Scientific) for 1 h at room temperature. The distribution of fluorescence in SM sections was analyzed using a fluorescence microscope (Axiovert 200®, Zeiss, Jena, Germany).
7
Immunohistochemical Evaluation of Vascular Markers
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Immunohistochemical staining was performed on 5-μm para n sections by using immunoperoxidase visualization. After routine depara nization, heat-induced epitope retrieval was performed through immersion of the slides in 0.01 M sodium citrate buffer (pH 6.0). To block the endogenous peroxidase activity and nonspeci c binding of antibodies, the sections were preincubated for 1 h at room temperature in 0.1 M PBS containing 10% normal goat serum and 0.3% H 2 O 2 . The sections were then incubated for 20 h at 4°C with rabbit polyclonal anti-vWF antibody (1:100; Abcam, Cambridge, Massachusetts, USA) or rabbit polyclonal anti-VEGF antibody (1:50; Santa Cruz Biotechnology) as primary antibodies. The sections were then treated for 1 h at 37°C with biotinylated goat antimouse or antirabbit IgG (1:200, Jackson ImmunoResesarch Laboratories Inc.). Following the reaction produced using reagents from an avidin-biotin complex kit (Vector Laboratories, Inc.), the reaction products were visualized using a diaminobenzidine substrate kit (Vector Laboratories, Inc.) according to the recommendations of the manufacturer.
Lung cytokines IL-1β, TNF-α, and MIP-2 levels were determined using the Bio-Plex multiplex assay system (Bio-Rad, Hercules, CA, USA) and Procarta immunoassay kit (Affymetrix) according to the manufacturers' protocols.
Lung cytokines IL-1β, TNF-α, and MIP-2 levels were determined using the Bio-Plex multiplex assay system (Bio-Rad, Hercules, CA, USA) and Procarta immunoassay kit (Affymetrix) according to the manufacturers' protocols.
8
CaMKII Signaling Pathway Analysis
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The isolated LV tissues or cultured cells were homogenized as described [18] . Primary antibodies used in this study include rabbit polyclonal anti-CaMKII antibody, rabbit polyclonal anti-phospho-CaMKII (Thr287) antibody, rabbit monoclonal anti-GAPDH antibody (Cell signaling technology), rabbit polyclonal anti-VEGF antibody (Santa Cruz Biotechnology) and rabbit polyclonal anti-CD31 antibody (Abcam). The second antibody used was goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology).
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