All samples were fixed in 10% buffered formalin phosphate, dehydrated and then embedded in paraffin. The blocks were sectioned at 6 μm thickness. For immunohistochemistry (IHC), deparaffinized and rehydrated sections were incubated with primary antibodies against human collagen type II (1:200), osteocalcin and TGF-β3 (1:200) (Abcam, Cambridge, MA) at 4°C overnight, followed by incubation with appropriate secondary antibodies. Immunostaining was carried out using the Vectastain ABC kit and NovaRED peroxidase substrate kit (Vector Labs, Burlingame, CA, USA). Images were acquired with the OLYMPUS CKX41 microscope.
Tgf β3
Manufactured by Abcam
Sourced in United States
TGF-β3 is a recombinant human protein that belongs to the transforming growth factor beta (TGF-β) superfamily. It is a cytokine involved in the regulation of cell growth, differentiation, and other cellular functions.
Automatically generated - may contain errors
Lab products found in correlation
Tgf β1, by Abcam (5 mentions)
Tgf β2, by Abcam (3 mentions)
Novared peroxidase substrate kit, by Vector Laboratories (2 mentions)
Vectastain abc kit, by Vector Laboratories (2 mentions)
3 3 diaminobenzidine (dab), by Merck Group (2 mentions)
Ckx41 microscope, by Olympus (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Horseradish peroxidase conjugated anti igg, by Santa Cruz Biotechnology (1 mentions)
Un scan it gel version 5, by Silk Scientific (1 mentions)
Gapdh, by Boster Bio (1 mentions)
Vimentin, by Boster Bio (1 mentions)
α sma, by Boster Bio (1 mentions)
N cadherin, by Santa Cruz Biotechnology (1 mentions)
Claudin 11, by Santa Cruz Biotechnology (1 mentions)
Occludin, by Abcam (1 mentions)
Alexa fluor, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Confocal microscope, by Olympus (1 mentions)
Smad3, by Novus Biologicals (1 mentions)
11 protocols using tgf β3
1
Immunohistochemical Analysis of Cartilage
2
Western Blot Analysis of Lung Proteins
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Total lung lysates were prepared by homogenizing lung tissues in Laemmli buffer. Proteins were separated by SDS-PAGE and electroblotted onto Immobilon-P membranes. Western blotting analyses were carried out as previously described32 (link). The antibodies used in this study included: TGF-β1, TGF-β3 (Abcam), fibronectin (Sigma-Aldrich) and α-smooth muscle actin (SMA) (Millipore). Secondary antibody was horseradish peroxidase-conjugated anti-IgG (Santa Cruz Biotechnology). Signals were visualized using SuperSignal West Dura Extended Duration Substrate (Pierce). The relative amount of proteins was quantified using gel analysis software UNSCAN-IT gel version 5.3 (Silk Scientific, Orem, UT), and normalized to β-actin internal loading control.
3
Immunoblotting Analysis of Cytoskeletal Proteins
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Protein was extracted and separated by SDS-polyacrylamide gel electrophoresis and was then transferred to polyvinylidene ethylene membranes, blocked from the nonspecific antibody binding sites with non-fat milk powder (concentration of 0.5%) at room temperature for 2 h, and then polyclonal antibodies against α-SMA (1 : 200, Boster, China), vimentin (1 : 200, Boster, China), TGF-β1 (1 : 200, BioVision, USA), TGF-β3 (1 : 100, Abcam, USA), and GAPDH (1 : 5000, Boster, China) were added. This was followed by incubation overnight at 4°C, followed by incubation with a secondary antibody (1 : 5000) for 2 h, and then autoradiograms were taken with ECL reagent in the chamber. Finally, imaging analysis was carried out by an automated electrophoresis gel imaging system.
4
Immunohistochemical analysis of cell junctions
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Histological sections (5 μm) were deparaffinized in xylene and rehydrated in 100, 95, 80, and 70% ethanol. Antigen retrieval was performed with pH 6.0 buffer citrate and endogenous peroxidase blocking with 0.3 % H2O2 in PBS. Sections were incubated overnight in 1% bovine serum albumin in PBS at room temperature. Primary antibodies (claudin-11 and N-cadherin, both 1/200 dilution: Santa Cruz, Dallas, USA; occludin and TGF-β3, 1/50 and 1/100 dilution, respectively: Abcam, Cambridge, UK) were incubated overnight at 4°C in a moisturized chamber and the day after peroxidase-conjugated secondary antibody (1/50 dilution; Pierce anti-rabbit, anti-goat, and anti-mouse, Cambridge, UK) was added and reaction visualized with 3,3′-Diaminobenzidine (Sigma–Aldrich, Milan, Italy). Counterstaining was performed with haematoxylin alone. Negative control slices were tested using PBS instead of primary antibody.
5
Immunofluorescence Staining of Extracellular Matrix Proteins
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The histological sections and adherent cells were fixed with 4% paraformaldehyde (Sigma) for 15 min and washed twice with PBS. The sections and cells were blocked with 10% normal goat serum (NGS) for 30 min and incubated with mouse primary monoclonal antibody Decorin (DCN) (5 μg/ml), mouse primary monoclonal antibody TGF-β1 (5 μg/ml), rabbit primary polyclonal antibodies fibromodulin (FMOD) (5 μg/ml), rabbit primary polyclonal antibodies TGF-β3 (5 μg/ml, Abcam, Cambridge, MA, USA), and human nuclear antigen (HNA) primary antibodies for overnight at 4 °C. This was followed by incubation with goat anti-mouse and goat anti-rabbit secondary antibodies (Alexa Fluor, Invitrogen) for 1 h. The sections and cells were then washed with PBS, stained with 4′-6-diamidino-2-phenylindole (DAPI; 0.5 μg/ml) (Invitrogen) for 5 min at room temperature (RT) and photographed using a confocal microscope (Olympus, Tokyo, Japan).
6
Molecular Mechanisms in Bone Cell Regulation
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Primary antibodies include VEGF (Cat# sc‐80436, RRID:AB_1131207, Santa Cruz Biotechnology), OPN (Cat# sc‐10593, RRID:AB_2270967, Santa Cruz Biotechnology), ALP (Cat# sc‐365765, RRID:AB_10842161, Santa Cruz Biotechnology), OCN (Cat# ab13418, Abcam), Runx2 (Cat# MBS186093, RRID:AB_10888180, MyBioSource), TGF‐β3 (Cat# ab90264, RRID:AB_2050388, Abcam), Smad3 (Cat#NBP1‐88732, RRID:AB_11034477, Novus), Wnt3 (Cat# ab32249, Abcam), β‐catenin (Cat# 26170, RRID:AB_2629235, NewEast Biosciences) and GAPDH (Cat# JM‐3777‐100, RRID:AB_843142, MBL International). LNA anti–miR‐29b oligonucleotides (antagomiR‐29b) and control LNA–scrambled oligonucleotides were custom generated and provided by GenePharma. As a negative control (NC), 10 point mutations were made in the miR‐29b mature sequence generating an RNA sequence that is not expressed in the rat genome.
7
Histological Evaluation of Skin Grafts
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Paraffin-embedded tissues from the skin grafts were sectioned at thickness of five micrometers and then mounted on adhesive glass slides. Hematoxylin and eosin (H&E) staining was performed according to standard procedures. Moreover, deparaffinized sections of day 14 were subjected to Masson's trichrome and picrosirius red staining to observe the collagen frameworks.
For immunohistochemistry analysis, deparaffinized sections of day 14 were incubated with VEGF polyclonal antibody (Abcam) and TGF-β3 (Abcam), respectively, at 4°C overnight and then followed by biotinylated goat anti-rabbit IgG antibody (Invitrogen) with PBS for 1 h. At last, visualization was performed with DAB (Sigma). Quantitative evaluations of TGF-β3 and collagen I and collagen III were assessed using Image Pro Plus 6.0 software (USA) according to mean density (IOD/area).
For immunohistochemistry analysis, deparaffinized sections of day 14 were incubated with VEGF polyclonal antibody (Abcam) and TGF-β3 (Abcam), respectively, at 4°C overnight and then followed by biotinylated goat anti-rabbit IgG antibody (Invitrogen) with PBS for 1 h. At last, visualization was performed with DAB (Sigma). Quantitative evaluations of TGF-β3 and collagen I and collagen III were assessed using Image Pro Plus 6.0 software (USA) according to mean density (IOD/area).
8
Quantifying TGFβ Isoforms in Cell Lysates
9
Immunofluorescence Analysis of TGF-β Isoforms in Corneal Samples
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Rat eyes were enucleated, embedded in Tissue-Tek O.C.T. compound, and frozen in liquid nitrogen. Sections (6 μm thick) were cut and mounted to polylysine-coated glass slides. NL and diseased human corneas obtained from the Midwest Eye Bank were embedded in Tissue-Tek O.C.T. compound immediately upon arrival. The cryostat sections of wounded and healed organ-cultured human corneas of diabetic and age-controlled groups were a gift from Dr. Alexander V. Ljubimov, Cedars-Sinai Medical Center (2 (link)). After drying in air, followed by a 10-min fixation in 4% paraformaldehyde, slides were blocked with 2% BSA in PBS for 1 h at room temperature. Sections were then incubated with rabbit primary antibody (TGF-β1, TGF-β2, and TGF-β3 1:50, Abcam). This was followed by fluorescein isothiocyanate anti-rabbit antibody (1:100; Jackson ImmunoResearch Laboratories). Slides were mounted with Vectashield mounting medium containing DAPI and examined under a Carl Zeiss fluorescence microscope Axioplan 2 equipped with an ApoTome digital camera or using confocal microscopy (TCSSP2; Leica). Controls were similarly treated, but the primary antibody was replaced with rat or rabbit IgG.
10
Transwell Assay for TGF-beta Signaling
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The Transwell chamber was purchased from Corning company (USA). Propidium iodide (PI) for the cell cycle was purchased from Sigma Aldrich (St Louis, MO, USA). CCK8 was purchased from Dojindo Molecular Technologies (Rockville, MD, USA). For Western blotting, the antibodies p-Smad2/3, p-Smad4, N-cadherin, E-cadherin, and Vimentin were purchased from Cell Signaling (MA, USA), while the LTBP4, TGFβ1, TGFβ2, TGFβ3, ARG1, and NOS2 antibodies were purchased from Abcam (MA, USA). LAP (latency-associated peptide) antibody was purchased from Thermo Fisher Scientific (MA, USA). The total and active TGFβ1 ELISA kits were purchased from BioLegend (CA, USA). The mass cytometry (CyTOF) antibodies were purchased from Fluidigm (CA, USA). The CD11B, LY6G, and LY6C antibodies were purchased from BioLegend (CA, USA). IgG-IP beads were purchased from Sigma Aldrich (MO, USA).
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