Tumor sections were handled as described previously 9 (link). Sections were incubated with anti-mouse LYVE-1 polyclonal (Ab 14817, 1:200; Abcam, Cambridge, MA, USA) or monoclonal anti-α-smooth muscle actin Sigma (αSMA A2547, 1:1000; Sigma, France), and rat monoclonal anti-mouse CD31 (clone MEC 13.3, 1:1000; BD Pharmingen, Franklin Lakes, NJ, USA) antibodies.
αsma a2547
Manufactured by Merck Group
Sourced in United States
αSMA A2547 is a laboratory reagent produced by Merck Group. It is an antibody that specifically binds to alpha-smooth muscle actin, a protein found in various cell types. The product is intended for use in research applications involving the detection and analysis of alpha-smooth muscle actin expression.
Automatically generated - may contain errors
Lab products found in correlation
Ab14917, by Abcam (4 mentions)
Triton x 100, by Merck Group (2 mentions)
Alexa 488 or 546, by Thermo Fisher Scientific (2 mentions)
Glass bottom dish, by AGC Techno Glass (2 mentions)
Cd31 clone mec 13, by BD (2 mentions)
Anti mouse cd31 clone mec 13, by BD (1 mentions)
Protein block serum free, by Agilent Technologies (1 mentions)
Hrp conjugated streptavidin, by Agilent Technologies (1 mentions)
Biotinylated secondary anti rabbit igg antibodies, by Agilent Technologies (1 mentions)
Dm750 photomicroscope, by Leica (1 mentions)
Mab4304, by Merck Group (1 mentions)
Bz x700 microscope, by Keyence (1 mentions)
Sc 1265, by Santa Cruz Biotechnology (1 mentions)
A5786, by ABclonal (1 mentions)
A11534, by ABclonal (1 mentions)
Collagen 1, by ABclonal (1 mentions)
Ha tag 3724, by Cell Signaling Technology (1 mentions)
N ethylmaleimide, by Merck Group (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Col1a1 sc 293182, by Santa Cruz Biotechnology (1 mentions)
16 protocols using αsma a2547
1
Immunohistochemical Staining of Mouse Tissues
2
Quantifying α-SMA Expression in Kidney Sections
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Immunohistochemistry (IHC) staining was performed as previously described28 (link). Briefly, tissues were fixed in 10% formalin for 36-h and embedded in paraffin or frozen-embedded in OCT solution (Tissue-Tek). Paraffin sections were prepared at a 4-μm thickness and mounted on microscope slides (Trajan Scientific and Medical, VIC, Australia). Antigen retrieval was performed at 99 °C for 20 min in 0.01 M citric buffer, pH 6.0. Endogenous peroxidase was deactivated with 3% H2O2 (Sigma-Aldrich, Dublin, Ireland). The slides were then blocked by Protein Block Serum-Free (Dako, Glostrup, Denmark), and incubated with α-SMA (A2547, 1:20,000, Sigma-Aldrich, MIS, USA,) at 4 °C overnight. After washing with TBST for 5 min, the slides were incubated with biotinylated secondary anti-rabbit IgG antibodies (Dako) for 30 min. The slides were washed again with TBST before staining with HRP-conjugated streptavidin (Dako) for 10 min. Using a light microscope (Leica DM750 photomicroscope with ICC50W digital camera), six consecutive non-overlapping fields from each kidney section were photographed and the percentage of staining area was quantitated using the Image J software (National Institutes of Health, USA).
3
Immunofluorescence Staining of Stem Cells
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Cells were cultured in a glass-bottom dish (AGC Techno Glass, Shizuoka, Japan) and fixed with 4% paraformaldehyde for 20 min at 4 °C before being permeabilization with 0.1% Triton X-100 (Sigma) for 10 min at 25 °C. After blocking with 5% normal goat serum in DPBS for 30 min at RT, samples were incubated with primary antibodies at 4 °C overnight. After washing with DPBS, the samples were incubated with secondary antibodies conjugated to Alexa 488 or 546 (Thermo Fisher Scientific) for 30 min at 25 °C. After washing with DPBS, mounting medium containing DAPI was used. The following primary antibodies were used: anti-βIII tubulin (TUJ1; G712A, Promega, Madison, Wisconsin, USA), α-smooth muscle cell actin (α-SMA; A2547, Sigma), SOX17 (MAB1924; R&D Systems), OCT3/4 (SC5729, Santa Cruz Biotechnology, Santa Cruz, California, USA), NANOG (RCAB004P–F, ReproCELL, Kanagawa, Japan), SSEA4 (MAB4304, Merck Millipore, Billerica, Massachusetts, USA), and TRA-1-60 (MAB4360, Merck Millipore). Images were acquired with a BZ-X700 microscope (Keyence). All antibodies, except for the anti-TUJ1 antibody (1:300), were used at a 1:150 dilution in 5% normal goat serum.
4
Immunohistochemical Analysis of Tissue Samples
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The tissue sections were fixed with acetone for 5 min and air-dried for 10 min. The sections were then immersed in 0.3% H2O2/PBS for 5 min to remove peroxidase activity. After treating with blocking solution for 1 h, primary antibodies were applied and incubated for 1 h, followed by secondary antibodies (HRP-conjugated goat anti-rabbit/mouse IgG), reacting for another hour. The sections were then stained with DAB for 5 min and rinsed with distilled water for another 10 min. The primary antibodies used in this experiment were TGF-β (sc-146, Santa Cruz), α-SMA (A2547, Sigma-Aldrich), Collagen I (A5786, ABclonal), IL-6 (sc-1265, Santa Cruz), IL-17A (A12454, ABclonal), and TNF-α (A11534, ABclonal).
5
Protein Immunoprecipitation and Western Blot Analysis
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Protein extracts for immunoprecipitation were prepared using nondenaturing immunoprecipitation buffer supplemented with protease and phosphatase inhibitor cocktails (78440, Thermo Fisher Scientific) and 20 mM N-ethylmaleimide (E3876, Sigma-Aldrich). Following protein quantifications, primary antibodies against His-Tag (12698, Cell Signaling Technology), Smad3 (sc-101154, Santa Cruz Biotechnology), or GSH (101-A, Virogen) were added to immunoprecipitate indicated proteins using Protein A Magnetic Beads (S1425S, New England Biolabs). The beads were washed, and protein elutes were analyzed by Western blotting. For Western blotting, cells or homogenized tissues were lysed in radioimmunoprecipitation assay (RIPA) buffer. Primary antibodies for Western blotting include GLRX (ab187507), Smad3 (ab28379), and TGFBR1 (ab235178) from Abcam; α-SMA (A2547) from Sigma-Aldrich; Timp-1 (sc-21734) and Col1a1 (sc-293182) from Santa Cruz Biotechnology; HA-Tag (3724), His-Tag (12698), and p-Smad3 (9520) from Cell Signaling Technology; and ZFYVE9 (PA5-67946) and TGFBR2 (PA5-36115) from Invitrogen. Following incubation with secondary antibodies, Pierce ECL Substrate was used for signal detection.
6
Immunofluorescence Analysis of Tumor Vasculature
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Tumour sections (5‐μm cryostat sections) were fixed in 4% paraformaldehyde for 10 min at room temperature and blocked in 1% horse serum in Tris‐buffered saline (TBS) for 1 h. The sections were then incubated overnight at 4 °C with anti‐rabbit LYVE‐1 polyclonal antibody (Ab14917, 1 : 200; Abcam, Cambridge, MA, USA) or mouse CD31 monoclonal antibody (clone MEC 13.3, 1 : 1000; BD Pharmingen, San Diego, CA, USA) and mouse anti‐α‐smooth muscle actin monoclonal antibody (αSMA A2547, 1 : 1000; Sigma) or anti‐Ki67 (ab16667, 1 : 500; Abcam). Preparations were mounted and analysed using a Leica microscope (Leica DMI4000B; Leica, Richmond, IL, USA) and counted at 10× magnification (CD31/αSMA) and 40× magnification (Ki67). The results are given as the number of vessels per mm2 of sections.
7
Immunoblotting Protein Expression Analysis
8
Fibrosis-related Protein Detection Assay
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PAI-1 (614024) and fibronectin (610077) antibodies were obtained from BD Biosciences (Franklin Lakes, NJ, USA). Phospho-Smad3 (#9520) and Smad2/3 (#3102) antibodies were provided by Cell Signaling (Danvers, MA, USA). CTGF antibodies (SC-101586) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and TGF-β1 antibody (NBP1-80289) was obtained from Novus Biologicals (Littleton, CO). Horseradish peroxidase-conjugated goat anti-rabbit (G21234) and anti-mouse (G21040) antibodies were purchased from Invitrogen (Carlsbad, CA, USA). α-SMA (A2547) and β-actin (A5441) antibodies, MTT (M2128), and dimethylsulfoxide (D8418) were acquired from Sigma Chemicals (St. Louis, MO, USA). TGF-β (240-B) was purchased from R&D Systems (Minneapolis, MN, USA). CTGF antibody (sc-101586) obtained from Santa Cruz Biotechnology (Santa Cruz, CA). CCl4 (33650-0330) was received from Junsei Chemical (Kyoto, CO).
9
Immunofluorescence Staining of Stem Cells
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Cells were cultured in a glass-bottom dish (AGC TECHNO GLASS, Shizuoka, Japan) and fixed with 4% paraformaldehyde for 10 min at 4 °C before being permeabilized with 0.1% Triton X-100 (Sigma) for 10 min at room temperature (RT). After blocking with 5% normal goat serum in Gibco™ Dulbecco's phosphate-buffered saline (DPBS; Thermo Fisher Scientific) for 30 min at RT, samples were incubated with primary antibodies at 4 °C overnight. After washing with DPBS, samples were incubated with secondary antibodies conjugated to Alexa 488 or 546 (Thermo Fisher Scientific) for 30 min at RT. After washing with DPBS, mounting medium with DAPI was used.
Primary antibodies specific for the following proteins were used in this study: OCT4 (C-10; Santa Cruz Biotechnology, Dallas, TX), NANOG (ReproCell), Tra 1-60 (MAB4360; Sigma–Aldrich), SSEA4 (MAB1435, R&D Systems), KLF4 (ab216875; Abcam), PRDM14 (ab187881; Abcam), anti-βIII Tubulin (TUJ1, Promega, Madison, Wisconsin), α-smooth muscle cell actin (α-SMA; A2547; Sigma), SOX17 (MAB1924; R&D Systems). Images were acquired using an LSM510 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany). All antibodies, except for the anti-TUJ1 antibody (1:300), were used at a 1:150 dilution in 5% normal goat serum.
Primary antibodies specific for the following proteins were used in this study: OCT4 (C-10; Santa Cruz Biotechnology, Dallas, TX), NANOG (ReproCell), Tra 1-60 (MAB4360; Sigma–Aldrich), SSEA4 (MAB1435, R&D Systems), KLF4 (ab216875; Abcam), PRDM14 (ab187881; Abcam), anti-βIII Tubulin (TUJ1, Promega, Madison, Wisconsin), α-smooth muscle cell actin (α-SMA; A2547; Sigma), SOX17 (MAB1924; R&D Systems). Images were acquired using an LSM510 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany). All antibodies, except for the anti-TUJ1 antibody (1:300), were used at a 1:150 dilution in 5% normal goat serum.
10
Western Blot Analysis of Liver Proteins
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Liver tissue was homogenized in lysis buffer containing 15 mM 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propanesulfonate, 0.15 M NaCl, 2 mM ethylenediaminetetraacetic acid (pH 8), 1 mM phenylmethanesulfonyl fluoride, 1 mM Na3VO4, and Tris-Cl (pH 7.5). Western blot analysis was performed as described previously (17 (link),30 (link)) using primary antibodies specific for goat polyclonal anti-human HB-EGF, goat polyclonal anti-human HGF (AB-294-NA; R&D Systems), mouse monoclonal anti-mouse α-smooth muscle actin (α-SMA; A2547; Sigma-Aldrich), rabbit polyclonal anti-mouse Bcl-2 (sc-492; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit polyclonal anti-mouse Bcl-xL (sc-634; Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-mouse Bax (sc-7480; Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-mouse collagen I (ab88147; Abcam) or mouse monoclonal anti-mouse α-tubulin (T6074; Sigma-Aldrich). The densities of the bands were measured, and the signal ratios of α-SMA/α-tubulin and collagen I/α-tubulin were calculated. For detection of each molecule, 25 µg of protein was loaded (40 µg in the case of α-SMA). Band densities were measured using ImageJ software.
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