We prepared 4 μm thick sections for each group of tumor tissues. The sections were labeled using anti-Ang-1, anti-Tie-2, anti-SCF, and anti-Flt-3L primary antibodies (Abcam; at 1:200, 1:50, 1:200, 1:180, respectively) at 4°C overnight and then incubated with a secondary antibody (goat anti-rabbit IgG; Zhongshan Golden Bridge, Beijing, China) for 1 h at room temperature. Following DAB coloration, hematoxylin counterstaining, dehydration and clearing in xylene, the slides were mounted. Staining intensity and positive area were analyzed by the average density (Average Optical Density, AOD) using image J software (V1.8.0.172). AOD =Integral Optical Density (IOD)/Positive area.
Anti scf
Manufactured by Abcam
Sourced in China, United States
Anti-SCF is a laboratory reagent used in research applications. It functions as an antibody that specifically binds to and detects the presence of Stem Cell Factor (SCF) protein in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti ang 1, by Abcam (1 mentions)
Goat anti rabbit igg, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Enhanced chemiluminescence reagent, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Cytiva (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Odyssey application software v1, by LI COR (1 mentions)
Peroxidase conjugated goat anti rabbit igg, by Beyotime (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Protease inhibitor cocktail, by Beyotime (1 mentions)
4 protocols using anti scf
1
Quantitative Analysis of Angiogenic Factors
2
Western Blot Analysis of SCF Protein Expression
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Proteins from Hepa1-6, EL4, Hep G2, 3B, SK, and Huh7 cells were separated on 12% polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Amersham Biosciences, Uppsala, Sweden). These blots were incubated for 1 h at room temperature in Tris-buffered saline containing Tween (TBST; 20 mM Tris–Cl, 140 mM NaCl, pH 7.5, 0.05% Tween 20) also containing 5% skim milk. Primary antibody used was anti-SCF (diluted 1:1,000, Abcam, Cambridge, MA, USA). Blots were incubated with primary antibodies overnight at 4 °C. After washing three times in TBST, blots were incubated with horseradish peroxidase-conjugated secondary antibody (diluted 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at room temperature. Immunoreactive complexes were visualized using enhanced chemiluminescence reagents (Millipore, Billerica, MA, USA).
3
Immunocytochemistry and Immunohistochemistry Protocols for Melasma
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For immunocytochemistry, cells were fixed in 4% paraformaldehyde for 10 min at room temperature and permeabilized with 0.2% Triton X-100. The non-specific binding of the antibody was blocked with 1% bovine serum albumin (BSA) for 1 h, followed by overnight incubation of cells with anti-SCF (Abcam) antibody at 4 °C. Immunohistochemical staining was performed on 4% paraformaldehyde-fixed, paraffin-embedded sections. Antibodies against CD31 (1:75 dilution; Novocastra, Newcastle, UK) and SCF (1:200 dilution; Abcam) were used for protein detection. Informed written consent was obtained from three melasma women patients (average age 43.3 years) before the skin biopsies. The study was approved by the institutional review board of Ajou University Hospital (AJIRB-DEV-DE3-15-491) and conducted in accordance with the approved human ethics guidelines. All participants were informed of the instructions and procedure of experiments and signed written informed consent forms prior to the experiments.
4
Western Blot Analysis of SCF in Murine Retina
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Eye samples were prepared after mice with euthanized. Retinas were then isolated and homogenized in an ice-cold mixture of RIPA buffer (Beyotime, China) containing protease inhibitor cocktail (Beyotime). Extracts were separated using 12% sodium dodecyl sulfate poly-acrylamide gels and transferred onto polyvinylidene fluoride membranes. Membranes were incubated in TBST (12.5 mM Tris–HCl, pH 7.6, 75 mM NaCl, 0.1% Tween 20) containing 5% fat-free milk for 1 h at room temperature, then transferred into solution containing primary antibodies at 4°C overnight, and probed with indicated secondary antibodies in TBST for 2 h at room temperature. Membranes were exposed on an Odyssey infrared imaging system with the Odyssey Application software V1.2.15 (LI-COR Biosciences, United States). All blots were analyzed by ImageJ (National Institutes of Health, United States). The relative levels of SCF were determined by normalizing against β-actin. The primary antibodies used were as follows: anti-SCF at 1:1000 (Cat# ab64677; Abcam), anti-β-actin at 1:1000 (Cat# ab179467; Abcam). The secondary antibody used was peroxidase-conjugated goat anti-rabbit IgG at 1:2000 (Beyotime).
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