Four young (AG11747, AG11242, AG10046, AG05415) and four aged (AG04064, AG04383, AG04152, AG4057) HFB cell lines that were used for the proliferation assay were plated in 5% DMEM in 6 well plates for 6 hours and then serum deprived overnight with 1% DMEM. The following day cells were exposed to saline or lidocaine with or without TGF-β1. All media were changed to 1% DMEM after 24 hours of exposure and cell lysates were collected after 48 hours. Cells were lysed in NP40 buffer with protease and phosphatase inhibitors (Sigma, Saint Louis, MO). Lysates were prepared for electrophoresis as described (29 (link)). Equal amounts of protein were subjected to 10% SDS-PAGE, followed by transfer to nitrocellulose. After incubation with 5% milk, blots were probed with antibodies (1-5μg/ml) against COL III (ABCAM, Cambridge, MA), COL I (ABCAM, Cambridge, MA) and GAPDH (Millipore, Billerica, MA).
Col 3
Manufactured by Abcam
Sourced in United Kingdom
Col-III is a collagen type III antibody that can be used for the detection and quantification of collagen type III in various sample types. It is a tool for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Col 1, by Abcam (7 mentions)
α sma, by Abcam (3 mentions)
Extl3, by Abcam (2 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
Np 40 buffer, by Merck Group (1 mentions)
Gapdh, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Boster Bio (1 mentions)
Il 17a, by Boster Bio (1 mentions)
Il 10, by Abcam (1 mentions)
Tnf α, by Boster Bio (1 mentions)
Interleukin 6 (il 6), by Boster Bio (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Anti tgf β, by Abcam (1 mentions)
Anti cd11c, by Abcam (1 mentions)
Anti ifn γ, by Abcam (1 mentions)
Anti cd45, by Abcam (1 mentions)
Anti relmα, by Abcam (1 mentions)
Anti sdf 1, by Abcam (1 mentions)
Smad2 3, by Cell Signaling Technology (1 mentions)
Smad7, by Cell Signaling Technology (1 mentions)
9 protocols using col 3
1
Evaluating Collagen Expression in HFB Cells
2
Immunofluorescence Analysis of ISCs
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3
Protein Extraction and Quantification
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The total protein is extracted from tissue or cells for 30 min with 50 mL lysis buffer (RIPA, Beyotime) containing 0.5 mL protease inhibitors (PMSF, Boster, China). The total protein concentration is quantified by BCA kit. Primary antibodies against to col I (1 : 1000, Abcam, UK), col III (1 : 1000, Abcam, UK), α-SMA (1: 1000, CST, USA), CD9, CD63 (1 : 1000, Proteintech, China), IL-6 (1 : 1000, Boster, China), IL-17A (1 : 1000, Boster, China), TNF-α (1 : 1000, Boster, China), IL-10 (1 : 1000, Abcam), TNF-α (1 : 1000, Boster, China), RORϒt (1 : 1000, Boster, China) and Foxp (1 : 1000, Boster, China), and GAPDH (1 : 1000, Zhuangzhi, China) are used for the analysis. The intensity of protein expression and normalized against GAPDH is analyzed by ImageJ software.
4
Comprehensive Wound Healing Evaluation
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The wound bed tissues were harvested at 3, 7, 14, 21, and 28 days after surgery, and the two cell sheet group tissues were fixed with 4% paraformaldehyde and then embedded in paraffin and cut into 5-mm thick longitudinal sections. The sections were subjected to hematoxylin and eosin (H&E) staining for histological analysis of wound regeneration. In addition, Masson staining was carried out to reveal the collagen accumulation state. For immunofluorescence staining, to investigate the in vivo immune reaction of curcumin treatment in the different groups, anti-CD45, anti-CD11c, anti-IFN-γ, anti-Relmα, anti-SDF1, anti-TGFβ, and anti-IL1RN antibodies (Abcam) were used to identify lymphocytes, M1 macrophages, TH1 cells, M2 macrophages, stromal cell-derived factor 1 (SDF1)+ cells, transforming growth factor (TGF)β, and IL-1 receptor antagonist (IL1RN). To investigate the contents of the cell sheets, sections were incubated with antibodies against type I collagen (Col-I, 1:50, Abcam) and type III collagen (Col-III, 1:80, Abcam). Secondary antibodies were goat anti-rabbit IgG (Alex Fluor488 conjugated, 1:200, CST) and goat anti-rat IgG (Alex Fluor555 conjugated, 1:500, CST). All data were analyzed using ImageJ software.
5
Investigating Intestinal Stem Cell Signaling
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ISCs were seeded into Nunclon™ 35mm petri dishes and either cultured alone, or in the presence of shRNA, or rhReg1, or U0126 (10μM, Sigma, CA, USA), or LY294002 (10μM, Sigma, CA, USA), or SB431542 (10μM, Selleck, Houston, USA) for 24 hours unless otherwise specified. After 24 hours, ISCs were lysed with ice-cold lysis buffer supplemented with protease inhibitors (Roche, Basel, Switzerland). After protein content determination and separation with 12%(w/v) SDS-PAGE, Western blotting was performed, as described previously [25 (link)], using antibody against primary antibody in 2.5% non-fat dried milk in Tris-buffered saline with Tween-20 (TBST) buffer. The primary antibody were as follows: Reg1 (1:3000, Abcam, Cambridge, UK), EXTL3 (1:200, Santacruz, Dallas, USA), α-SMA (1:1000, Abcam, Cambridge, UK), Col-I (1:5000, Abcam, Cambridge, UK), Col-III (1:5000, Abcam, Cambridge, UK), FN (1:5000, Abcam, Cambridge, UK), TGF-β, Akt, P-Akt, Erk, P-Erk, Smad2/3, P-Smad2/3, Smad7 (1:1000, Cell Signaling, MA, USA), β-Actin (1:2000, Sigma, CA, USA).
6
Western Blot Analysis of Smad2, Col I, and Col III
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Radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) was used to collect the proteins from cells, and bicinchoninic acid protein assay was then conducted to quantify the protein concentration. Equal amount of protein (40 µg/lane) was separated by 12% SDS-PAGE and then transferred to polyvinylidene difluoride membranes. Next, the membranes were blocked with 5% non-fat milk at room temperature for 1.5 h, incubated with primary antibodies: Smad2 (1:1,000; cat. no. 5339; Cell Signaling Technology, Inc.), Col I (1:1,000; cat. no. ab34710; Abcam), Col III (1:1,000; cat. no. Ab7778; Abcam) and β-actin (1:1,000; cat. no. 4970; Cell Signaling Technology, Inc.) at 4˚C overnight. Subsequently, membranes were incubated with anti-rabbit horseradish peroxidase-conjugated immunoglobulin G secondary antibody (cat. no. 7074; 1:2,000; Cell Signaling Technology, Inc.) at room temperature for 2 h. At the end of the experiment, an enhanced chemiluminescence detection system (Applygen Technologies, Inc., Beijing, China) was used to observe the protein bands. For densitometry detection, analysis with ImageJ 1.38X software (National Institutes of Health, Bethesda, MD, USA) was performed.
7
Western Blotting of Extracellular Matrix Proteins
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Western blotting analysis was performed with the adherent cell treated with releasing buffer for 24 h. Briefly, equal amounts of protein in the cell lysates were subjected to electrophoresis on 7% of polyacrylamide gel electrophoresis (SDS-PAGE) gel at 110 V, followed by transferring to poly (vinylidene fluoride) (PVDF) membranes. After blocked with 5% of albumin from bovine serum (BSA) for 2 h at room temperature, the membranes were incubated with antibodies of Col I (Rabbit, 1:3000; Abcam, Cambridge, UK), Col III (Rabbit, 1:1000; Abcam, Cambridge, UK), α-SMA (Rabbit, 1:1000; Abcam, Cambridge, UK), TGF-β1(Rabbit, 1:1000; Abcam, Cambridge, UK), and β-actin antibodies (Goat, 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). After being washed four times with Tris-Buffered Saline Tween-20 (TBST), the immunoreactive traces were detected by an enhanced chemiluminescence (ECL) detection kit (Amersham Biosciences, Chalfont St. Giles, UK). The intensity of protein expression on the membranes was analyzed by Image J software.
8
Spinal Cord Injury Histology
9
Cardiac Fibroblast Protein Expression Analysis
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Collected hearts and cardiac fibroblasts were lysed using RIPA protein extraction reagent with protease inhibitors (Beyotime, Shanghai, China). Then, equal amounts of proteins were run on 8–15% gels and blotted onto PVDF membranes. Subsequently, membranes were blocked with 5% non-fat dried milk in TBST and incubated with primary antibodies overnight at 4°C. The primary antibodies were Col III, Col I, MMP-2, TLR4, TGF-β (1:1000, Abcam, United States), Smad3, p-Smad3 (1:1000, Cell Signaling Technology, Inc.) and GAPDH (1:5000, Bioworld Technology, Inc.), as a loading control. After washing twice, membranes were incubated with appropriate HRP-conjugated secondary antibodies for 2 h at room temperature. Signals were detected using an ECL chromogenic substrate and quantified with densitometry by Quantity One software (Bio-Rad, Berkeley, CA, United States).
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