Proteins were isolated from cultured cells or mouse skin tissue at the injured site. Specific protein levels were detected by anti-PLOD1, anti-ROS, anti-α-SMA, anti-TGF-β1, anti-collagen I ELISA kits (R&D, Carpinteria, CA, USA).
Anti tgf β1
Manufactured by R&D Systems
Sourced in United States
Anti-TGF-β1 is a laboratory reagent that can be used for the detection and quantification of transforming growth factor beta 1 (TGF-β1) in various biological samples. It is a specific antibody that binds to TGF-β1 and can be used in techniques such as ELISA, Western blotting, and immunohistochemistry.
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Lab products found in correlation
Anti α sma, by R&D Systems (1 mentions)
Alexa flour 488, by Thermo Fisher Scientific (1 mentions)
Alexa flour 594, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Eclipse ti u, by Nikon (1 mentions)
Anti pecam 1, by Abcam (1 mentions)
Anti f4 80, by Santa Cruz Biotechnology (1 mentions)
Pparα, by Abcam (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Anti tgf β1 antibody, by R&D Systems (1 mentions)
Tgf β1, by R&D Systems (1 mentions)
Anti il 1β, by Santa Cruz Biotechnology (1 mentions)
Anti tnf α, by Abcam (1 mentions)
Envision system, by Agilent Technologies (1 mentions)
Anti fibronectin, by Abcam (1 mentions)
Anti il 1β, by R&D Systems (1 mentions)
β actin, by R&D Systems (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
9 protocols using anti tgf β1
1
Protein Analysis in Skin Injury
2
Anti-TGF-β1 Antibody Effects on Myoblast Implantation
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Animals were treated systemically by i.p. injection of the specific blocking antibody anti-TGF-β1 (Clone 9016, R&D Systems) in PBS with 0.5% BSA (100 μg/animal/dose) at the time of myoblast implantation (day 0) and after 2, 4, and 6 days, according to the previously published treatment schedule (Wan et al, 2008 (link)). IgG1 antibody in PBS with 0.5% BSA (100 μg/animal/dose; R&D Systems) was given i.p. as control.
3
Histological Examination of Liver Tissue
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Liver specimens were fixed overnight in 10% formalin solution, then dehydrated, embedded in paraffin, and cut into 10 μm sections. A cross-section taken from the blocks was stained with hematoxylin and eosin stains (H&E, Sigma-Aldrich, St. Louis, MO, USA) and Masson’s trichrome. For immunohistochemistry, sections were incubated with anti-TGF-β1 (R&D Systems, Minneapolis, MN, USA), psmad2/3 and pJNK (Cell signaling) for 1 h at 37 °C, processed by an indirect immuneperoxidase technique using a commercial kit (DAKO, CA, USA). Light microscopy was used to acquire the immunohistochemical images. The primary antibodies for the cells and tissue section immunofluorescent staining are as follows: E-cadherin and vimentin were purchased form Cell signaling, and secondary antibodies conjugated with Alexa Flour 488 and Alexa Flour 594 were purchased from Invitrogen. Cells were counterstained with Hoechst 33342 (ImmunoChemistry, Bloomington, MN, USA). Slides were mounted using ProLong® Gold antifade reagent (Molecular Probes® by Life Technologies™, Carlsbad, CA, USA). Fluorescence micrographs were acquired with a fluorescence microscope (Nikon ECLIPSE Ti-U, Nikon Co., Tokyo, Japan).
4
Immunohistochemical Evaluation of Fibrosis, Angiogenesis, and Oxidative Stress
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We performed immunohistochemistry for TGF-β1, PECAM-1, 8-OH-dG, PPARα and the TUNEL assay. Four-µm-thick sections were incubated overnight in a humidified chamber at 4°C with anti-TGF-β1, a profibrotic growth factor (1∶100; R&D Systems, Minneapolis, MN); anti-PECAM-1, a mouse endothelial cell marker (1∶100; Abcam, Cambridge, UK); anti-F4/80, an inflammatory cell marker (1∶100; Santa Cruz Biotechnology, CA); the in situ TUNEL assay, an apoptotic marker (Chemicon-Millipore, Billerica, MA); 8-OH-dG, an oxidative DNA damage marker (1∶100; COSMO BIO, Tokyo, Japan); and PPARα (1∶200; Abcam, Cambridge, UK).
5
Quantification of Tube Formation and Cell Migration
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Tube formation assays were performed as described previously28 (link). Tube formation was monitored for 12–16 h under a phase-contrast microscope and quantified by counting the number of master junctions in four separate experiments in a blinded manner using ImageJ software. For Transwell migration assays, 1 × 105 HUVECs in 200 μl of serum-free M199 were seeded into the upper inserts (8-μm pores) of 12-well plates (Cat# 353182, Corning), and 600 μl of M199 supplemented with 20% FBS was added to the lower chamber. HUVECs or MCECs were then treated with 1 μg/ml PBS (control), 1 μg/ml LRG1, 5 ng/ml TGF-β1 (Cat# 240-B, R&D Systems), 10 μg/ml anti-TGF-β1 antibody (Cat# MAB1835, R&D Systems), and 5 ng/ml TGF-β1 + 10 μg/ml anti-TGF-β1 antibody (premixed overnight for neutralization). After 18 h of culture at 37 °C, non-migrated cells on the upper surface of the insert were removed with a cotton swab. Migrated cells were fixed and stained with crystal violet (Cat# V5265, Sigma) and then eluted with 10% acetic acid. The number of migrated or invaded cells was counted in four random fields.
6
Immunohistochemical Analysis of Inflammatory Markers
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Paraffin-embedded tissue sections at 4 μm thickness were deparaffinized with xylene, dehydrated in gradually decreasing concentrations of ethanol, and then treated with 3% hydrogen peroxidase in methanol for 10 min to block endogenous peroxidase activity. The tissue sections were immersed in a 10 mM sodium citrate buffer (pH 6.0) for 5 min at 95 °C. The last step was repeated using a fresh 10 mM sodium citrate solution (pH 6.0). The sections were allowed to remain in the same solution while cooling for 20 min, and they were then rinsed in phosphate-buffered saline (PBS). After, the sections were incubated with a primary antibody (1:100 dilution) for 1 h at 37 °C. The primary antibodies were follows: anti-TNF-α and anti-fibronectin (Abcam, Cambridge, MA, USA), anti-IL-1β (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-TGF-β1 (R&D Systems, Minneapolis, MN, USA). The signal was visualized using an Envision System (DAKO, Carpinteria, CA, USA) for 30 min at 37 °C. 3,3′-diaminobenzidine tetrahydrochloride (DAB) was used as the coloring reagent, and hematoxylin was used as the counter-stain.
7
Amygdalin Modulates Inflammatory Response
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Amygdalin (purity: High Performance Liquid Chromatography (HPLC) ≥ 98%, DST200710 004) was purchased from Lemeitian Pharmaceutical Technology Co., Ltd. (Chengdu, China). Lipopolysaccharide (LPS) was purchased from Sigma Chemical Co. (St. Louis, MO, United States). Reactive Oxygen Species Assay Kit (ROS, DCFH-DA) were obtained from Beijing Solarbio Technology Co., Ltd. (Beijing, China). Antibodies used herein including anti-IL6, anti-TNF-α, anti-p-AKT, anti-AKT, anti-SRC, anti-p-SRC, anti-VEGFA, anti-MAPK1, anti-EGFR, anti-IL-1β, anti-TGF-β1, β-actin, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG and Horseradish Peroxidase (HRP)-conjugated goat anti-mouse IgG polyclonal antibody were obtained from R&D Systems (Minnesota, USA).
8
Induction and Suppression of iTregs
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CD4+ T-cells were isolated from PBMCs of healthy donors through negative sorting using microbeads (Miltenyi Biotec Company, Bergisch Gladbach, North Rhine-Westphalia, Germany), according to the manufacturer’s instructions. Subsequently, the CD4+ T cells were cocultured with irradiated BC-derived cells with S1P1 overexpression or depletion in OKT3 (R&D, Minneapolis, MN, USA)-precoated 48-well plates for 5 days to induce the generation of (i)Tregs in the presence or absence of anti-IL-10, anti-TGF-β1 (5 ng/ml, R&D, Minneapolis, MN, USA) and FTY720 (0.1 μM, Sigma-Aldrich, St. Louis, MO, USA). The percentages of Treg cells were determined using FACS analysis. For the suppressive analysis, PBMCs from healthy donors were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE, 10 μM, eBioscience), and the CFSE-labeled PBMCs were plated onto OKT3-coated 96-well plates and cocultured with different (i)Tregs at a ratio of 10:1 or in medium alone for 5 days. The PBMCs were subsequently harvested and stained for CD4, CD25, and Foxp3, and staining data were acquired and detected through FACS analysis.
9
Transwell assay for Treg migration
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Isolated naïve T cells were seeded in six‐well plates with anti‐CD3 (0.5 μg/m:, ebioscience) and anti‐CD28 (2 μg/mL, ebioscience) stimulated for 2 days. Then, cocultured with NPC cells with transwell in the presence or absence of anti‐TGF‐β1 (10 ng/ml, R&D System) for 4 days.
Migration of Tregs was tested by 5 μm pore‐size transwell (Corning Costar). 1 × 104 Tregs were seeded in upper chamber with NPC cells supernatant in lower chamber and cultured with or without anti‐CCL20 (10 ng/mL, R&D System) for 24 hours. The number of migrated cells in the lower chamber was counted by microscope.
Migration of Tregs was tested by 5 μm pore‐size transwell (Corning Costar). 1 × 104 Tregs were seeded in upper chamber with NPC cells supernatant in lower chamber and cultured with or without anti‐CCL20 (10 ng/mL, R&D System) for 24 hours. The number of migrated cells in the lower chamber was counted by microscope.
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