After MRI scanning, all rats were sacrificed, dissected and examined for gross abnormalities. Macroscopical specimen of entire reproductive tracts were harvested from the rats and fixed in 10% formalin. The specimen was dissected into sections at 1-3 mm interval by L.W., with 10 years of experience in human and murine gynecological pathology. The tumors were analyzed for morphology, and then the tissues were embedded in paraffin and cut into 3-μm-thick slices. Hematoxylin-eosin (HE) staining was performed to observe the histological type. Immunohistochemical (IHC) (Primary antibodies of CD31, VEGF and Ki67 purchased from Cell Signaling Technology, CA, USA) was applied to investigate the MVD, expression of VEGF and Ki67 in positive cells in EOCs. MVD was analyzed in CD31-stained vascular endothelial cells and determined as the mean microvessel at 3 microscopic fields of vision (HPF, × 200). VEGF expression displayed as brownish yellow granules in the cytoplasm and intercellular spaces, and determined as the mean integrated optical density (IOD) measured by Image-Pro Plus image analysis software at 3 HPFs (×200). Ki67-positive expression indicated the appearance of yellow or brown granules in the nucleus and determined as the percentage of positive tumor cells in 1000 tumor cells assessed at 10 HPFs (×200) with 1000 random cells.
Vascular endothelial growth factor (vegf)
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, China
VEGF is a recombinant protein that functions as a growth factor. It is involved in the regulation of angiogenesis, the process of new blood vessel formation.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Cell Signaling Technology (19 mentions)
Gapdh, by Cell Signaling Technology (15 mentions)
Bcl 2, by Cell Signaling Technology (14 mentions)
Hif 1α, by Cell Signaling Technology (11 mentions)
P akt, by Cell Signaling Technology (10 mentions)
Mmp 9, by Cell Signaling Technology (10 mentions)
Caspase 3, by Cell Signaling Technology (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (7 mentions)
Cyclin d1, by Cell Signaling Technology (7 mentions)
Survivin, by Cell Signaling Technology (6 mentions)
Erk1 2, by Cell Signaling Technology (6 mentions)
Cleaved caspase 3, by Cell Signaling Technology (5 mentions)
β catenin, by Cell Signaling Technology (5 mentions)
E cadherin, by Cell Signaling Technology (5 mentions)
P erk1 2, by Cell Signaling Technology (5 mentions)
Vimentin, by Cell Signaling Technology (4 mentions)
Bca protein assay kit, by Beyotime (4 mentions)
Mcl 1, by Cell Signaling Technology (4 mentions)
Mmp 2, by Cell Signaling Technology (4 mentions)
86 protocols using vascular endothelial growth factor (vegf)
1
Histopathological Analysis of Rat Ovarian Tumors
2
Western Blot Analysis of Aortic Proteins
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Total proteins were extracted from the thoracic aortas by lysis buffer, and protein concentration was measured by BCA kit (TaKaRa, Kyoto, Japan). Protein samples (30 µg) were subjected to 10% SDS-PAGE and then transferred onto nitrocellulose membrane. Following blocking with 5% skim milk, the membranes were incubated with the primary antibody (ICAM-1, 1:1000; VCAM-1, 1:1000; ET-1, 1:1000; NF-κB p65, 1:1000; p-NF-κB p65, 1:1000, Abcam, Cambridge, MA, USA; VEGF, 1:1000; IκBα, 1:1000, Cell Signaling Technology, Beverly, MA, USA; GAPDH, 1:1000, Sino Biologial, Beijing, China) at 4℃ overnight. Afterwards, peroxidase-labeled secondary antibody (anti-rabbit IgG, 1:5000, Abcam) was used for incubation for 1 h at room temperature. Protein blots were visualized with an enhanced chemiluminescence kit. Finally, density of western blot bands was analyzed using Quantity One 1-D Analysis Software (Bio-Rad Laboratories).
3
Evaluation of Antimicrobial and Angiogenic Properties
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Methicillin-resistant Staphylococcus aureus (MRSA) and Gram-negative bacteria Escherichia coli (E. coli) were purchased from Guangdong Microbial Culture Collection (China). Dulbecco's modified Eagle's medium (DMEM), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and fetal bovine serum (FBS) were purchased from Gibco (USA). Human skin keratinocytes cells (HaCat) and human umbilical vein endothelial cells (HUVEC) were obtained from American type culture collection (ATCC). The 2,2’-bis(anthracene-9,10-diylbis(methylene))-dimalonic acid (ABDA) was supplied from Shanghai Civi Chemical Technology Co., Ltd. 1-methyl-2-pyrrolidone (NMP) was purchased from Aladdin. The primary antibodies to CD31, VEGF, bFGF, Tubulin were obtained from Cell Signaling Technology (USA). We used deionized water throughout the experiment.
4
Wogonin's Anti-Cancer Potential Explored
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A2780 cells were purchased from Sigma-Aldrich Co. (St Louis, MO) and were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco). Cells were tested with a Cell Culture Contamination Detection Kit (Thermo Fisher Scientific) and results appeared negative for mycoplasma contamination. Wogonin, with a chemical structure shown in Figure 1(a) , was purchased from Aokebio (Beijing, China). Methylpiperidinopyrazole (MPP) was purchased from Apexbio. The antibodies were from Abcam (Akt, β-actin), Proteintech (caspase-3, cleaved-caspase-3, cyclin D1, CDK4, and CDK6), Santa Cruz Biotechnology (ER-α), and Cell Signaling Technology (Bcl-2, Bax, VEGF, and p53).
5
Western Blot Analysis of Protein Expression
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25μg of total protein lysates were separated on 12% polyacrylamide SDS gels and electroblotted on nitrocellulose membranes as previously described [47 (link)]. Briefly, after blocking with 5% non-fat milk in TBS-T (20 mM Tris, 137 mM NaCl, 0.1% Tween-20, pH 7.6), the membrane was probed with one of the following primary antibodies (at 1:1000 dilution) at 4°C overnight: rabbit monoclonal antibodies against P4HB, total p44/42 MAP kinase (Erk 1/2) (total MAPK), phosphor-p44/42 MAP kinase (Erk1/2) (Thr202/Tyr204) (pMAPK), and VEGF (all from Cell Signaling Technology Inc.). The membranes were washed three times with TBS/T followed by incubation with 1:10,000 diluted HRP-conjugated secondary antibodies (Invitrogen-Zymed Laboratories) at 4°C for 1 hour. Signal on blots was developed using ECL detection system (GE Biosciences, Buckinghamshire, England).
6
Evaluating Endothelial Cell Proliferation
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To determine the effects of EMS and miR-126-5p on EC proliferation, an immunofluorescence assay was performed. Briefly, sections were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 5 min at room temperature, permeabilized, and blocked for 30 min with 0.1% Triton X-100 and 1% bovine serum albumin. Subsequently, the fixed sections were washed and incubated for 1 h with primary antibodies against vWF (1:200; Cell Signaling Technology, Shanghai, China), VEGF (1:200; Cell Signaling Technology), CD31 (1:100; Santa Cruz Biotechnology, Dallas, TX, USA), or eNOS (1:200; Cell Signaling Technology) overnight at 4°C. The sections were then washed three times with PBS and incubated with fluorescence-conjugated species-specific secondary antibodies. The samples were subsequently counterstained with 4’,6-diamidino-2-phenylindole (DAPI, Beyotime Biotechnology, Shanghai, China) for identification of the nuclei. Photographs were acquired using a fluorescence microscope. The quantities of vWF-, VEGF- and CD31-positive cells indicated the proliferation and distribution of capillary ECs. The quantities of eNOS-positive cells indicated the repair and proliferation of capillaries. For each group, the numbers of positively stained cells in five random visual fields (100 μm × 100 μm) were counted. All the experiments were performed in triplicate.
7
Nanomedicine Formulation Development
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Gefitinib, simvastatin, and fluorescent dyes were obtained from Melone Pharmaceutical (Dalian, China). Osimertinib was from Selleck Chemicals (Texas, USA), and murine cytokines were from Peprotech (New Jersey, USA), and LPS from Sigma-Aldrich (St. Louis, USA). Soybean phosphatidylcholine (SPC), cholesterol, and DSPE-PEG2000, DSPE-PEG-NHS, and DSPE-PEG-Mal were obtained from Advanced Vehicle Technology (Shanghai, China). The derivative T12 peptide (sequence: CGGGTHRPPMWSPVWP) was synthesized by Bankpeptide Biological Technology (Hefei, China). The primary antibodies of EGFR, phospho-EGFR (Tyr1068), Erk1/2, phosphor-Erk1/2 (Thr202/Tyr204), Akt, phosphor-Akt (Ser473), VEGFR2, VEGF, caspase3, cleaved-caspase3, LC3, TGF-β, GAPDH, and galectin-3 were purchased from Cell Signal Technology (Boston, USA). The primary antibody β-Actin was obtained from Sigma-Aldrich (St. Louis, USA). The primary antibodies of CD206 and TNF-α were from Abcam (Cambridge, UK). Anti-HIF-1α was obtained from Novus (Shanghai, China) and anti-IFN-γ was from Absin (Shanghai, China). The horseradish peroxidase (HRP)-conjugated goat anti-rabbit/mouse lgG secondary antibody was obtained from Beyotime (Shanghai, China). All other reagents (analytical grade) were provided by Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China).
8
Evaluating Growth Factor Expression on Stent
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WB was performed to confirm growth factor expression, as previously described13 (link),16 (link). Transfected cells (5 × 105) were seeded onto the stent material for 24 h and treated with Dox (5 μg/ml) for 2 days. The conditioned medium was collected and precipitated using trichloroacetic acid (TCA, Sigma, USA). The pellet was dissolved in 200 µl of RIPA buffer (Thermo, IL, USA). SDS-PAGE was performed using a 1% acrylamide gel. HGF (R&D Systems, MN, USA) and VEGF (Cell Signaling, MA, USA) antibodies were used at a 1:1000 dilution.
9
Western Blot Analysis of Protein Markers
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According to the manufacturer’s protocol, RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) was employed to extract protein from cells. Protein concentrations were measured using a BCA Protein Assay Kit obtained from Beyotime Institute of Biotechnology Co., Ltd. (Shanghai, China). Next, 10% dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was employed to separate the proteins, which were subsequently transferred onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore) through electro-blotting. Then, 5% skim milk was used to block the membranes for 1 hour in tris-buffered saline with Tween (TBST), after which the membranes were incubated with primary antibody rabbit anti-ATG7 antibody (1:1,000, #8558, Cell Signaling), anti-p21 antibody (1:1,000, #2947, Cell Signaling), anti-Survivin (1:1,000, #2808, Cell Signaling), anti-E-cadherin (1:1,000, #3195, Cell Signaling), N-cadherin (1:1,000, #13116, Cell Signaling), VEGF (1:1,000, #9698, Cell Signaling), and Vimentin (1:1,000, #5741, Cell Signaling). After incubation overnight at 4 °C, the membranes were incubated for a second time with goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies for 1 hour at 37 °C. The band densities were analyzed with an automatic digital gel image analysis system (Bio-Rad CFX-96, CA, USA).
10
Histopathological and Immunohistochemical Analysis of Tumors
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Tumors were harvested and fixed with 4% formaldehyde at 4 °C for 24 h. Tissues were then embedded using paraffin and sectioned serially. The slides were dewaxed and stained with H&E for histopathological analysis. For IHC analysis, paraffin sections were deparaffinized, rehydrated, and subjected to antigen retrieval. Slides were then blocked with 10% normal goat serum and incubated with anti-Ki67 (1:500, Abcam, Cambridge, MA, USA), anti-CD31 antibody (1:100, Thermo Fisher Scientific), PTEN (1:100, Cell Signaling Technology, Beverly, MA, USA), or VEGF (1:100, Cell Signaling Technology) at 4 °C overnight, followed by an incubation with biotinylated secondary antibody and streptavidin-HRP. Immunoreactivity was visualized using AEC solution (Thermo Fisher Scientific).
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