Mouse liver tissues were fixed in 10% zinc formalin, embedded in paraffin, sectioned at 4 μm, and subjected to H&E staining and immunostaining as described [22] (link). Monoclonal rat anti-GRP94 (1:200) is from Enzo Life Sciences (Farmingdale, NY). Cell proliferation was evaluated by Ki67 staining (SP-6, 1:50, Thermo Scientific, Waltham, MA). Monoclonal mouse anti-glutamine synthetase (GS) (1:400, BD Biosciences, San Jose, CA) was used to examine liver zonation. Monoclonal mouse anti-hepatocyte paraffin 1 (HepPar1) (1:25, DakoCytomation, Denmark A/S) and polyclonal rabbit anti-wide spectrum cytokeratin (panCK) (1:75, Abcam, Cambridge, MA) antibodies were used to identify hepatocytes and cholangiocytes/LPCs, respectively. Mesenchymal cells were detected by monoclonal mouse anti-α-SMA (1:2000, Sigma, St. Louis, MO). Monoclonal rat anti-CD34 (1:100) is from BioLegend (San Diego, CA).
Mouse monoclonal anti α sma
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany
Mouse monoclonal anti-α-SMA is a primary antibody that binds to alpha-smooth muscle actin (α-SMA), a cytoskeletal protein found in various cell types, including smooth muscle cells and myofibroblasts. This product can be used in research applications for the detection and localization of α-SMA in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1, by Zeiss (3 mentions)
Monoclonal rat anti grp94, by Enzo Life Sciences (1 mentions)
Prolong dapi, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Quanta 200, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Enhanced chemiluminescence detection system, by Cytiva (1 mentions)
β tubulin, by Cell Signaling Technology (1 mentions)
Tetramethylbenzidine, by Merck Group (1 mentions)
Sb203580, by Corning (1 mentions)
Tgf β1, by Corning (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
Horseradish peroxidase, by Thermo Fisher Scientific (1 mentions)
Rabbit anti β actin, by Cell Signaling Technology (1 mentions)
Tetramethylbenzidine tmb, by Merck Group (1 mentions)
17 protocols using mouse monoclonal anti α sma
1
Immunostaining of Mouse Liver Tissue
2
Immunofluorescence Analysis of NLRP3, TLR4, IL-1β, IL-6, and α-SMA
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Cells were fixed with 4% paraformaldehyde. After serum blocking for 30 min, cells were incubated with the primary antibody to polyclonal rabbit anti-NLRP3 (Sigma, St. Louis, U.S.A.); polyclonal rabbit anti-TLR4 (Santa Cruz Biotechnology, Dallas, U.S.A.); polyclonal rabbit anti-IL-1β (Santa Cruz Biotechnology, Dallas, U.S.A.); monoclonal mouse IL-6 (Abcam, Cambridge, United Kingdom); or monoclonal mouse anti-α-SMA (Sigma, St. Louis, U.S.A.), then with rhodamine- or FITC-conjugated secondary antibody (Life Technologies, Carlsbad, EUA). Finally, cells were counterstained with DAPI and mounted on glass slides. PTC profiles containing fluorescent material were expressed as a percentage of the total number of nuclei. Multiple images were taken per group and the mean fluorescence intensity (MFI) of the stainings was quantitated using ImageJ software and calculated in relation to the number of nuclei per field.
3
Fibroblast-to-Myofibroblast Differentiation Assay
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To assess the effect of TA-65 on fibroblast-to-myofibroblast differentiation, MPLF or MEF cells seeded on Lab-Tek chamber slides were treated with recombinant TGF-ß1 (10 ng/mL, Sigma T7039, France) for 96 h in presence or absence of TA-65 (2 μM) or Imetelstat (1 μM). After a first wash, cells were fixed with 4% paraformaldehyde at 4 °C for 10 min, then permeabilized with 0.1% Triton X-100 during 15 min at room temperature, washed and blocked with 2% bovine serum albumin in Dulbecco’s phosphate-buffered saline at room temperature for 30 min. Slides were subsequently incubated with monoclonal mouse anti-α-SMA (1:200, Sigma A5228) in a humidified chamber overnight at 4 °C. The fluorescent signal was detected using a goat anti-mouse secondary antibody (1:500, conjugated with Alexa 488, Life technologies). Slides were mounted using ProLong DAPI (Life Technologies). A fluorescence microscope coupled to a digital camera utilizing the Axiovision® software was used to view and acquire images (Zeiss, Jena, Germany). The quantification of signal was performed by using Image J software.
4
Comprehensive Tissue Analysis Protocol
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Tissue samples were fixed in 10% formalin, embedded in paraffin, and 5-μm-thick sections were obtained. Samples were stained with Hematoxylin and Eosin (H&E), or Masson trichrome. Other tissue samples were fixed for 30 min with 2.5% glutaraldehyde in phosphate buffer (pH 7.2). After fixation, the samples were dehydrated in a graded ethanol series. The air-dried samples were sputter coated with platinum palladium, and examined with a scanning electron microscope (SEM, Quanta 200; FEI). Immunochemical staining was performed by staining frozen cross-sections of samples fixed in cold acetone with mouse monoclonal anti-αSMA (1:100, Sigma Aldrich), and rabbit monoclonal anti-CD31 primary antibodies (1:100; Abcam), followed by incubation with the appropriate HRP-linked secondary antibody, and visualization with DAB.
5
Western Blot Analysis of Cytoskeletal and ECM Proteins
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The protein concentrations of the cell lysates were determined by BCA assay. Equal amounts of proteins (20 μg) from cell lysates were resolved by 8–16% SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham Biosciences, Pittsburgh, USA). After blocking with 5% milk, the membranes were incubated with primary antibodies overnight at 4 °C followed by incubation with horseradish peroxidase-conjugated secondary antibodies and detection by use of an enhanced chemiluminescence detection system (Amersham Bioscience, Pittsburgh, USA). Primary antibodies included mouse monoclonal anti-α-SMA (1:10000 dilution, Sigma-Aldrich, St. Louis, USA); rabbit polyclonal anti-MMP-1, MMP-3 and MMP-7 (1:1000 dilution, Sigma-Aldrich, St. Louis, USA); and β-tubulin (1:1000 dilution; Cell Signaling Technology, Danvers, USA). Bands were digitalized, and their densities were quantified using image analysis software. The results are expressed as a ratio of band density to total β-tubulin density.
6
Quantifying Myofibroblast Markers in HBFs
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Cells were cultured in 96-well plates for 24 h in a complete medium, which was changed for a serum-free medium without or with SB203580 (10 µM) alone or in combination with TGF-β1 (5 ng/mL; Corning, NY, USA). After five days of culture, the HBFs were fixed and permeabilized with ice-cold methanol and blocked with a 1% BSA/PBS solution with 0.1% Tween20/PBS. The HBFs were incubated overnight at 4 °C with primary antibodies in a 1% BSA/PBS solution (mouse monoclonal anti-α-SMA; rabbit polyclonal anti-fibronectin; rabbit polyclonal anti-Cx43; rabbit polyclonal anti-collagen1; all from Sigma-Aldrich, St. Louis, MO, USA). After triple rinsing, the plates were incubated with goat anti-mouse or goat anti-rabbit secondary antibodies conjugated with horseradish peroxidase (HRP) in a 1% BSA/PBS solution at room temperature for 1 h. Colorimetric reactions were induced by the addition of tetramethylbenzidine (Sigma-Aldrich, St. Louis, MO, USA) and stopped using 1N HCl/H2O. Absorbances were measured using MultiskanFC at 450 nm.
7
Immunofluorescence Analysis of Epithelial-Mesenchymal Transition
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NRK-52E monolayers were fixed in methanol (20 min, −20 °C), and blocked with 2% bovine serum albumin (BSA) in PBS (1 h, room temperature). Cells were then incubated with primary antibodies against epithelial cell marker rabbit moloclonal anti-E-cadherin (1:50, Abcam, Cambridge, UK) and mesenchymal marker mouse monoclonal anti-α-SMA (1:100; Sigma-Aldrich) with 2% BSA overnight at 4 °C). Secondary fluorescent conjugated anti-rabbit Alexa Fluor®647 (1:400; Invitrogen) was used for E-cadherin and anti-mouse Alexa Fluor®546 (1:400; Invitrogen) was used for α-SMA for 40 min at room temperature in dark. Slides were mounted using ProLong® Gold Antifade reagent with DAPI (Life technologies). Images were photographed using Olympus FV1000 Confocal microscope at ×40 magnification.
8
Western Blot Analysis of α-SMA Expression
9
Quantifying Cytoskeletal Proteins in HBFs
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Protein content analyses were performed in methanol-fixed HBFs using an in-cell ELISA assay according to a previously described protocol [40 (link)]. Cells were incubated overnight at 4 °C with the following primary antibodies: mouse monoclonal anti-α-SMA, mouse monoclonal anti-vinculin (from Sigma-Aldrich, St. Louis, MO, USA), and rabbit monoclonal anti-talin (from Cell Signaling Technology, Danvers, MA, USA), in a dilution of 1:2000. After triple rinsing, goat anti-mouse or goat anti-rabbit secondary antibodies conjugated with horseradish peroxidase (HRP; Thermo Fisher Scientific, Waltham, MA, USA) were added and incubated at room temperature for 1 h. Then, tetramethylbenzidine (TMB, Sigma-Aldrich, St. Louis, MO, USA) was used for the induction of colorimetric reactions, which were stopped afterward using 1 N HCl/H2O. Absorbances were detected using a MultiskanFC microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at λ = 450 nm.
10
Histological Analysis of Tissue Formation
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To determine IEL and IT formation, paraffin tissue sections were stained with elastica van Gieson, as recommended by the manufacturer (Muto Pure Chemicals, Tokyo, Japan). Immunohistochemical analysis was performed as described previously [8 (link), 9 (link)]. Rabbit polyclonal anti-t-PA (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit polyclonal anti-von Willebrand factor (Dako Cytomation, Santa Clara, CA, USA), mouse monoclonal anti-α-SMA (Sigma-Aldrich, St. Louis, MO, USA), rabbit polyclonal anti-MMP2 (Novus Biologicals, Little, CO, USA) antibodies were used.
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