The proteins of primarily cultured NSCs or spinal cord tissues from SCI rats were isolated by RIPA lysis buffer (Beyotime, Shanghai, China), and the concentrations were determined by a BCA detection kit (Beyotime). About 20ug proteins were loaded and electrophoresed onto 12% SDS polyacrylamide gel (GE Healthcare Bioscience, Marlborough, MA, United States) and then transferred to polyvinylidene fluoride (PVDF) membrane (MilliporeSigma, Burlington, MA, United States). The membranes were blocked by 5% non-fat milk for 60 mins at 37°C and were then incubated with anti-NGF monoclonal antibody (1:1000, Abcam, Cambridge, MA, United States), anti-CREB (1:1000, Abcam), anti-GDNF (1:500, Abclonal, Wuhan, China), anti-BDNF (1:1000, Abcam), anti-VEGF (1:1000, Boster, Pleasanton, CA, United States) and anti-β-actin (1:5000, Boster) overnight at 4°C. The primary antibody incubation was followed by incubation with a secondary peroxidase-conjugated anti-rabbit (1:5000, Boster) or anti-mouse antibody (1:5000, Boster) at room temperature for 2 h. The detection of signal was conducted using Bryo ECL kit (Bytotime), and the measurement of proteins’ expression was done by Image J (Joonas “Regalis” Rikkonen, Version 1.8.0).
Anti vegf
Manufactured by Boster Bio
Sourced in China
Anti-VEGF is a laboratory reagent used for the detection and quantification of vascular endothelial growth factor (VEGF) in various sample types. It functions by specifically binding to VEGF, which is a key regulator of angiogenesis, the process of new blood vessel formation.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Anti creb, by Abcam (1 mentions)
Anti β actin, by Boster Bio (1 mentions)
Anti mouse antibody, by Boster Bio (1 mentions)
Anti bdnf, by Abcam (1 mentions)
Bca detection kit, by Beyotime (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Anti cd31, by Boster Bio (1 mentions)
Anti cdk6, by Proteintech (1 mentions)
Anti ki67, by Proteintech (1 mentions)
Anti mmp2, by Proteintech (1 mentions)
Anti mmp9, by Proteintech (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Anti cyclin d1, by Proteintech (1 mentions)
Ecl plus reagent, by Beyotime (1 mentions)
Anti tgfβ1, by Boster Bio (1 mentions)
Sds page gel, by Beyotime (1 mentions)
Anti ilk, by Abcam (1 mentions)
Anti cox2, by Proteintech (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
5 protocols using anti vegf
1
Western Blot Analysis of Neural Factors
2
Immunohistochemical Analysis of Fracture Callus
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The anti-NGF/P75 (Boster Biological Technology, cat#: M01187), anti-VEGF (Boster Biological Technology, cat#: P802), and anti-CD31 (Boster Biological Technology, cat#: BM4213) were used for immunohistochemistry for measuring corresponded proteins expressions at the callus tissue between fractures. To block the endogenous peroxidase, H2O2 was added to the section after antigen repair and incubated for 10 minutes at room temperature and then washed with PBS buffer for 3 minutes three times and incubated with 100 µL of the blocking normal calf serum working solution for 15 minutes at room temperature. Then, it was incubated with 100 µL of primary antibody overnight at 4°C after removing the serum. On the second day, it was washed with PBS buffer for 3 minutes three times after being incubated at room temperature for 15 minutes. Then, it was incubated with100 µL biotin-labeled goat anti-mouse/rabbit IgG polymer at room temperature for 13 minutes and washed with PBS buffer for 3 minutes three times. It was then incubated with 100 µL of horseradish peroxidase-labeled streptolysin working solution at room temperature for10 minutes and washed with PBS buffer for 3 minutes three times. Appropriate amount of 3,3′-Diaminobenzidine (DAB) was freshly prepared and incubated for 5–8 minutes. Hematoxylin and eosin stain was incubated for 20 seconds and tap water was used for counterstaining.
3
Evaluating Tumour Biomarkers by IHC
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According to the manufacturer's instructions, the tumour tissues were first dewaxed and rehydrated using xylene and ethanol. Then, high-pressure heat was used for antigen retrieval. Next, anti-CDK6, anti-cyclin D1, anti-MMP-2, anti-MMP-9, anti-Ki67 (Proteintech, Wuhan, China), anti-VEGF (Boster) and anti-E-cadherin (CST, Danvers, MA, USA) were added, respectively. Finally, all sections were dehydrated, cleared, mounted and visualised with a diaminobenzidine-based colorimetric method and analysed with the Image Pro Plus 6.0 software (Media Cybernetics Inc.). The immunohistochemistry (IHC) index was defined as the average integral optical density.
4
Protein Expression Analysis in hESCs
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Proteins were extracted from cultured HESCs using RIPA lysis buffer, and the concentration of protein was detected by the BCA Protein Assay Kit (Beyotime). Samples were then separated by 10% SDS-PAGE gels (Beyotime). The primary antibodies were applied according to the provided recommendations: anti-ILK (1:5000, Abcam), anti-TGFβ1 (1:200, Boster), anti-SMAD2 (1:1000, Affinity), anti-VEGF (1:200, Boster), anti-COX-2 (1:700, Proteintech Group), anti-MMP-9 (1:800, Abcam) and anti-GAPDH (1:1000, Abcam). Finally, positive bands were detected using the chemiluminescent ECL Plus reagent (Beyotime) according to the manufacturer’s protocol. The densitometry of bands was quantified using Quantity One version 4.6.0 software (Bio-Rad). Expressions of proteins were normalized to GAPDH protein.
5
Western Blotting for Protein Analysis
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A549 and H292 cells (1 × 106 cells/well) were seeded in six-well plates overnight and were then exposed to the different treatments at the indicated time points. Cell lysates and tumour tissue homogenates were separated by SDS–PAGE and then transferred onto PVDF membranes (Millipore, USA). Next, WB was performed as previously described23 (link). The following specific primary antibodies were used: anti-phospho-STAT1 (1:1000, Tyr701, CST#9167), anti-STAT1 (1:1000, Tyr701, CST#9167), anti-IL-6 (1:1000, 21865-1-AP), anti-IL-8 (1:1000, sc-1265; Santa Cruz Biotechnology), anti-VEGF (1:1000, BA0407; Boster, China), and anti-GAPDH (1:5000, BL1039; Wuhan, China).
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