The live expression of Collagen I (COL1A1), Collagen III (COL3A1) and Tenascin-C (TNC) were visualized as previously described [50 (link)]. Briefly, four treatment groups (as described previously; control, FGF2, GDF5 and FGF2 + GDF5) of MSCs were seeded and cultured on the PCL scaffolds for 14 days. The scaffolds were washed with PBS (5 min.), fixed with 4% paraformaldehyde (Wako) for 20 minutes, and then blocked with 0.3% Triton-X (Sigma-Aldrich, St. Louis, MO, USA). After PBS washing, the scaffolds were incubated in 1% BSA (10 minutes; Fisher), then 10% normal goat serum (45 minutes at room temp.; Jackson ImmunoResearch), with PBS washing between each step. Primary antibodies (mouse; Collagen I (1:250, Sigma), Tenascin-C (1:250, Abcam), Collagen III (1:200, Abcam)) were then added to the respective wells and incubated overnight at 4°C. The secondary antibody (goat anti-mouse Cy3, Jackson ImmunoResearch) was added to each scaffold at a concentration of 1:500. Each scaffold was counterstained for 10 minutes at room temperature using DAPI (Sigma-Aldrich, St. Louis, MO, USA) at a 1:500 concentration. Images of each scaffold were then obtained using confocal microscopy (Zeiss LSM 780, Germany). Red pixels represent expression levels of each factor of interest, while blue represents the DAPI nuclear stain. Cy3 expression was quantified using Image J software (NIH).
Tenascin c
Manufactured by Abcam
Sourced in United States
Tenascin-C is an extracellular matrix glycoprotein that plays a role in cell adhesion and migration. It is involved in tissue remodeling and is expressed during development, wound healing, and pathological conditions such as cancer.
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Lab products found in correlation
P mlc2, by Cell Signaling Technology (4 mentions)
Total fak, by BD (4 mentions)
Py397 fak, by Thermo Fisher Scientific (4 mentions)
Fibronectin, by Abcam (3 mentions)
Gli 1, by Thermo Fisher Scientific (2 mentions)
Collagen 12a1, by Santa Cruz Biotechnology (2 mentions)
Integrin β1, by Merck Group (2 mentions)
Mabt409, by Thermo Fisher Scientific (2 mentions)
P mypt1, by Merck Group (2 mentions)
Collagen 3, by Abcam (2 mentions)
Total stat3, by Cell Signaling Technology (2 mentions)
Rock1, by Cell Signaling Technology (2 mentions)
Rock2, by Cell Signaling Technology (2 mentions)
P stat3, by Cell Signaling Technology (2 mentions)
P smad2 3, by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Vimentin, by Cell Signaling Technology (2 mentions)
Total mlc2, by Abcam (2 mentions)
Alexa fluor conjugated goat secondary anti mouse igg and anti rabbit igg antibodies, by Thermo Fisher Scientific (2 mentions)
Na934v, by GE Healthcare (2 mentions)
10 protocols using tenascin c
1
Visualizing Extracellular Matrix Proteins in MSCs
2
Immunohistochemistry Antibody Panel
3
Antibody Validation for Cellular Analysis
4
Western Blot Analysis of Fibroblast Proteins
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At termination of the experiments, fibroblasts were harvested and equal amounts of whole-cell lysates or culture media (15–20 μg) subjected to SDS-PAGE electrophoresis, as described (15 (link)). Membranes were incubated with primary antibodies specific for type I collagen (Southern Biotechnology, 1:1,000, 1310-01), ASMA (Sigma-Aldrich, 1:2,000, A5228), Fn-EDA (Sigma-Aldrich, 1:1,000, F6140), Tenascin-C (Abcam, 1:1,000, ab108930), phospho-Smad2 (Cell Signaling Technology, 1:400, 3108), or GAPDH (Santa Cruz Biotechnology Inc., 1:1,000, sc365062), and bands were detected using enhanced chemiluminescence (15 (link)). Band intensities were quantitated using ImageJ software (NIH) and corrected for GAPDH in each lane. See complete unedited blots in the supplemental material.
5
Immunohistochemistry Antibody Panel
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Antibodies were as follows: a-SMA (Sigma-Aldrich C6198, 1:1000), GLi-1 (Thermo Scientific PA5-32206, 1:500), Tenascin C (Abcam AB108930, 1:500), Fibronectin (Abcam AB2413, 1:500), Collagen 12A1 (Santa Cruz Biotechnology E-15 sc-68449, 1:200), Sox2 (Abcam ab97959, 1:500), Vimentin (Cell Signaling 5741S, 1:200), FAP (Abcam AB53066, 1:500), Collagen III (Abcam AB7778, 1:500), YAP (Cell signaling 4912, 1:200), β1integrin (EMD Millipore MABT409, 1:500), pY397-FAK (Invitrogen 44625, 1:200), total FAK (BD Biosciences 610088, 1:1,000), ROCK1 (Cell Signaling C8F7, 1:1,000), ROCK2 (Cell Signaling D1B1, 1:1,000), p-MLC2 (Cell Signaling 3671, 1:200), p-MyPT1 (Millipore ABS45, 1:200), p-Stat3 (Cell Signaling 9145, 1:200), p-SMAD2/3 (Cell Signaling 8828, 1:200), total Stat3 (Cell Signaling 9132, 1:1,000), GAPDH (Cell Signaling 2118, 1:5,000), CD45 (BD Biosciences 550539, 1:200), CD68 (Thermo Scientific Ab-3, 1:200), Alexa Fluor-conjugated goat secondary anti–mouse IgG and anti–rabbit IgG antibodies (Invitrogen A11012, 1:1,000 and A11005, 1:1,000) and HRP-conjugated rabbit secondary antibody (GE health care life sciences NA934VS, 1:5,000).
6
Tenascin-C Expression in Achilles Tendon Regeneration
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The treated Achilles tendon of each group was collected in week 1 and week 4 and performed frozen sections. All the sections were immune stained with Tenascin-C (1 : 100, Abcam) primary antibody, followed by Alexa Fluor 488 dye-labeled secondary antibody. DAPI (Roche) was used to stain cell nuclei and observed under the immunofluorescent microscope to check the cells transplantation regenerative processes.
7
Antibody Validation for Cellular Analysis
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Antibodies were as follows (used at 1μg ml−1 for western blotting and chromatin immunoprecipitation (ChIP) and at indicated concentrations for immunofluorescence studies): tenascin C (Abcam ab108930; 1:1,000), pY397-FAK (Invitrogen 44625; 1:200), total FAK (BD Biosciences 610088), pMLC2 (Cell Signaling 3671; 1:200), total MLC2 (Abcam ab92721, clone EPR3741; 1:200), p-MyPT1 (Millipore ABS45; 1:200), hyaluronic acid binding protein (Calbiochem, 385911; 1:500), aggrecan (Abcam ab3778; 1:500), versican (Abcam ab19345; 1:500), collagen 1 (Abcam ab34710; 1:1,000), propidium iodide (AcrosOrganics 440300250; 1 μg ml−1), β-actin (Sigma-Aldrich a5441), HIF1α (Abcam ab-1, for immunofluorescence; 1:200; Abcam ab1, for ChIP; Novus 100-449, for western blotting), hypoxyprobe (Hypoxyprobe, HP1-100 Kit; per manual), CD31 (BD 550389; 1:500), laminin (Abcam ab11575; 1:500), RNA polymerase II (Millipore 05-623B), rabbit IgG isotype control (Cell Signaling 2729; 1:500), Alexa Fluor-conjugated goat secondary anti-mouse IgG and anti-rabbit IgG antibodies (Invitrogen A11012 and A11005; 1:500) and HRP-conjugated rabbit secondary antibody (GE Healthcare Life Sciences NA934VS; 1:5,000).
8
Immunofluorescence Analysis of hACLF Cells
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hACLFs at P3 from the experimental and control groups were cultured on cover slips for 7, 10, and 14 days before the cell density was adjusted to 104 cells/ml. The cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% polyethylene glycol octylphenol ether (Triton X-100) for 10 minutes. The cells were then blocked with Lowenthal serum for 30 minutes. The following primary antibodies were incubated with the cells overnight at 4°C: anti-collagen I, collagen III, fibronectin, and tenascin-C (Abcam, Eugene). Secondary FITC- or CY3-labeled antibodies were then incubated with the samples for 1 hour. Cell nuclei were counterstained with 2-(4-amidinophenyl)-1H-indole-6-carboxamidine at room temperature for 5 minutes. Cover slips were slowly mounted onto slides with 90% glycerol and observed with an inverted fluorescence and phase-contrast microscope.
9
Immunohistochemical Analysis of Lung Tissue
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Lung samples were fixed in formaldehyde, embedded in paraffin and serial sections were prepared for cell (alpha-smooth muscle actin-α-sma cells) and ECM glycoprotein staining. Tissue sections were deparaffinized, rehydrated and then antigen was retrieved by being boiled in Tris-EDTA buffer (pH 9.0) for 5 min. Samples were blocked with 0,3% H2O2 for 1 h with avidin/biotin blocking kit (vector). Immunohistochemistry was carried out using primary antibody of tenascin C (1/300, Abcam), versican (1/250, Lifespan) and fibronectin domain EDA (1/400, Abcam), followed by biotin-avidin/peroxidase (Vectastain Elite ABC kit) incubation. Alpha-smooth muscle actin (α-sma) (RTU Flex, Dako) was performed with Autostainer Link Instrument, using EnVision Flex+, mouse, and high pH (Link) (Dako), according to the manufacturer’s recommendation. All sections were counterstained with haematoxylin.
10
Immunohistochemical Evaluation of Tissue
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Freshly isolated tissue was fixed in 4% paraformaldehyde and embedded in paraffin. Sections, 5 mm in thickness, were dewaxed and hydrated; endogenous peroxidase was blocked with 3% hydrogen peroxide for 10 min; and nonspecific binding was blocked with a protein blocker (DakoCytomation, Trappes, France). After antigen retrieval, sections were incubated in primary antibodies against a-SMA, tenascin-C (Abcam, Cambridge, MA, USA), CD163, and Reg3g (Antibodies-Online.com, Atlanta, GA, USA). EnVision + -HRP (DakoCytomation) was used as the secondary reagent for all immunostaining sections. The color reaction was developed with the NovaRED kit (Vector Laboratories, Burlingame, CA, USA) and counterstained with Mayer's hemalum.
TUNEL staining was performed using the In Situ Cell Death Detection Kit (Roche Applied Science), according to the manufacturer's instructions.
(continued from previous page) cell, LSP1 = leukocyte-specific protein 1, M1 = classically activated macrophage (type 1 macrophage), M2 = alternatively activated macrophage (type 2 macrophage), MMP = matrix metalloproteinase, MSC = mesenchymal stem cell, P0/P1 = passage 0 and 1, P4HA1 = prolyl 4hydroxylase subunit a1, Reg3g = regenerating islet-derived protein 3g, T reg = regulatory T cell
TUNEL staining was performed using the In Situ Cell Death Detection Kit (Roche Applied Science), according to the manufacturer's instructions.
(continued from previous page) cell, LSP1 = leukocyte-specific protein 1, M1 = classically activated macrophage (type 1 macrophage), M2 = alternatively activated macrophage (type 2 macrophage), MMP = matrix metalloproteinase, MSC = mesenchymal stem cell, P0/P1 = passage 0 and 1, P4HA1 = prolyl 4hydroxylase subunit a1, Reg3g = regenerating islet-derived protein 3g, T reg = regulatory T cell
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