After deparaffinization and hydration, sections were blocked with 3% hydrogen peroxide, followed by treatments with 10% normal goat serum (Cat. No. 16210072; Gibco; Thermo Fisher Scientific), primary antibodies α-SMA (A2547, MilliporeSigma), Collagen I (14695–1-AP, Proteintech), Collagen II (15943–1-AP, Proteintech), and HRP-conjugated goat anti-rabbit secondary antibody (1:400; Cat. No. A32731; Invitrogen). After DAB treatment, microscopic images were taken.
αsma a2547
Manufactured by Merck Group
22 citations
Sourced in United States
About the product
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.
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Market Availability & Pricing
The Monoclonal Anti-Actin, α-Smooth Muscle (clone 1A4) antibody, also known as αsma a2547, is currently commercialized by Sigma-Aldrich, a subsidiary of Merck KGaA, Darmstadt, Germany. The product is listed on Sigma-Aldrich's official website, indicating its active availability.
Pricing for the αsma a2547 antibody varies based on volume, with the following options:
- 100 μL: $523.00
- 0.2 mL: $631.12
- 0.5 mL: $1,206.00
These prices are sourced directly from Sigma-Aldrich's official product page.
Sigma-Aldrich also offers alternative antibodies targeting α-Smooth Muscle Actin, such as:
- Anti-Actin, α-Smooth Muscle antibody (Product No. A5228)
- Anti-Actin, α-Smooth Muscle - Cy3™ antibody (Product No. C6198)
- Anti-Actin, α-Smooth Muscle - FITC antibody (Product No. F3777)
These alternatives are also available through Sigma-Aldrich's official website.
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Sc 14939, by Santa Cruz Biotechnology (2 mentions)
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Check compatibility
22 protocols using «αsma a2547»
1
Immunohistochemical Analysis of Extracellular Matrix
Corresponding organizations : Ningxia Medical University General Hospital, Ningxia Medical University, Xianyang Normal University
2
Immunohistochemical Analysis of Tissue Samples
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).
Corresponding organizations : China Medical University, China Medical University Hospital, Chung Shan Medical University, National Taiwan University, Chung Shan Medical University Hospital
3
Immunofluorescence Analysis of Tumor Vasculature
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.
Corresponding organizations : Scientific Centre of Monaco, Inserm, Centre Antoine Lacassagne, Centre National de la Recherche Scientifique, Institut de Recherche sur le Cancer et le Vieillissement de Nice, Karolinska Institutet, Centre Hospitalier Universitaire de Nice, Centre Léon Bérard, Université Claude Bernard Lyon 1, Centre Hospitalier Universitaire de Bordeaux, International University of Monaco, Université Côte d'Azur
4
Immunofluorescence Staining of Stem Cells
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.
Corresponding organizations : National Center For Child Health and Development, Tokyo Medical and Dental University, The University of Tokyo, Saitama Medical University, University of Southern California
5
Quantifying α-SMA Expression in Kidney Sections
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).
Corresponding organizations : Royal North Shore Hospital, University of Sydney, Pharmaxis (Australia)
Top 5 most cited protocols using «αsma a2547»
1
Comprehensive Lung and Heart Histomorphometry
Lung and heart tissue was fixed in formalin and embedded in paraffin. Four micrometers thick paraffin-embedded tissue sections were deparaffinized and subsequently stained with hematoxylin and eosin (HE). In addition, lung tissue sections were immune-stained with anti-ED-1 (monocytes and macrophages; 1:5), anti-myeloperoxidase (MPO, RB-373-A1, Thermo Fisher Scientific, Fremont, CA, USA; diluted 1:1,500), anti-α smooth muscle actin (αSMA, A2547, Sigma-Aldrich, St. Louis, MO, USA; diluted 1:20,000), anti-von Willebrand factor (vWF, A0082, Dako Cytomation, Glostrup, Denmark; diluted 1:4,000), anti-collagen III (COL3A1, ab7778; Abcam; diluted 1:3,000), anti-pSMAD1 [diluted 1:2,000; this antibody cross-reacts with pSMAD5 and pSMAD8, which also act downstream of BMP type I receptors (Persson et al., 1998 (link); Rosendahl et al., 2002 (link))], anti-pSMAD2 (diluted 1:2,000), (Persson et al., 1998 (link); Rosendahl et al., 2001 (link)) and anti-transmembrane protein 100 (TMEM100, GTX83508; Gene Tex, Irvine, CA, USA; diluted 1:400), using the chromogenic substrate NovaRed or NovaRed and Vector SG Substrate on αSMA and vWF double stained sections, respectively (Vector, Burlingame, CA, USA), and counterstained briefly with hematoxylin using standard methods (de Visser et al., 2009 (link), 2010 (link)). Furthermore, elastin was visualized on Hart's stained lung sections (Simon et al., 2010 (link)). We used a Weibel type II ocular micrometer (Olympus, Zoeterwoude, The Netherlands) for morphometric analysis of the lung, (Wagenaar et al., 2004 (link)). Different (immuno)histochemically stained lung sections were used for each quantification. However, alveolar crests and pulmonary arteriolar wall thickness were determined on the same αSMA stained section. To exclude potential effects of heterogeneous alveolar development we investigated alveolar enlargement in experimental BPD in two different ways by studying mean linear intercept (MLI) and the number of alveolar crests. The MLI, determined on hematoxylin and eosin stained lung sections, was assessed in 10 non-overlapping fields at a 200x magnification for each animal (Dunnill, 1962 (link); de Visser et al., 2010 (link)). The quantification of alveolar crests is an indicator of the number of alveoli (Yi et al., 2004 (link)). The number of alveolar crests (Yi et al., 2004 (link)) was determined on αSMA-stained lung sections at a 400x magnification in 10 non-overlapping fields for each animal and were normalized to tissue and field. The number of MPO-positive neutrophilic granulocytes or ED-1 positive monocytes and macrophages was determined in the alveolar compartment and normalized to field and expressed as cells per mm2. Twenty fields in one section were studied at a 400x magnification for each experimental rat. HE-stained lung sections were used at a 400x magnification for alveolar septal thickness measurements by averaging 100 measurements per 10 representative fields. The number of vessels per field were counted at a 200x magnification in vWF-stained lung sections to determine capillary density. At least 10 representative fields per experimental animal were investigated. Results were expressed as relative number of vessels per mm2. Arteriolar wall thickness was assessed twice in elastin- or αSMA-stained lung sections at a 1,000x magnification by averaging at least 10 vessels with a diameter of <30 μm per rat for each of the two different staining methods. Medial wall thickness was calculated from the formula “percent wall thickness = 100” (Koppel et al., 1994 (link)). Muscularization of small arterioles (<50 μm) was determined on αSMA and vWF double stained lung sections, using the 50% αSMA layer circumference as a cutoff at a 400x magnification by counting 50 blood vessels per lung section (Alapati et al., 2011 (link); Chen et al., 2011 (link)). We excluded fields with large blood vessels or bronchioli from the analysis. Right and left ventricular free wall thickness was assessed at a 40x magnification in a transversal HE-stained section taken halfway the long axis by averaging 6 measurements per structure. For each heart RVH was calculated by dividing average RV free wall thickness and average LV free wall thickness. For morphometric studies in lung and heart, 6–8 and 10 rat pups per experimental group were studied, respectively. NIH Image J software was used for quantitative morphometry. Two independent researchers blinded to the treatment strategy performed the analysis (Yi et al., 2004 (link); de Visser et al., 2010 (link); Chen et al., 2016 (link)).
Corresponding organizations : Leiden University Medical Center, UCLA Medical Center, The Lundquist Institute, Harbor–UCLA Medical Center, Utrecht University, Papworth Hospital, University of Cambridge
2
Immunohistochemical Validation of Antibody Specificity
Muscle cryosections were immunostained in batched sets by the same individual for CCN2 (sc14939, Santa Cruz Biotechnology, 1:1000 dilution in PBS), collagen type 1 (ab6308, AbCam, Cambridge, MA, 1:300 dilution in PBS), alpha smooth muscle actin (αSMA, A2547, dilution 1:500, Sigma), phosphorylated extracellular signal‐regulated kinase (pERK, 4370, 1:200 dilution in PBS, Cell Signaling, Danvers, MA), or with an anti‐human IgG tagged with Dylight 650 (ab96906, Abcam) to detect the FG‐3019 antibody, using previously described methods.4 , 19 Sections were blocked with 5% goat serum and were then incubated with primary antibodies in moist incubation chambers overnight at room temperature. Appropriate secondary antibodies were diluted 1:100 in PBS and incubated on sections for 2 hours before washing in PBS. DAPI was used as a nuclear stain before coverslipping with 80% glycerol in PBS.
Specificity of the CCN2 and collagen type I antibodies used for immuohistochemistry was determined using western blots to check if the antibodies detected bands at the correct molecular weights. Membranes were incubated with primary antibodies overnight at 4°C. For CCN2, gels were made as described earlier for CCN1 and 3. For collagen type I, 8% gels were made without SDS in the gel, yet with SDS in sample and loading buffers, as described.38 The antibodies used detected bands at the correct molecular weights (Figure S1 A,B).
Preabsorption antibody‐protein blocking steps were also performed to demonstrate either the CCN2 or collagen type 1 specificity using CCN2 recombinant protein (CXT‐687, Prospec, East Brunswick, NJ) and collagen type 1 rat protein (C7661, Sigma). A two‐ to threefold excess of purified protein, respectively, was preincubated with the matching antibody overnight at 4°C, the mixture centrifuged, and the preabsorbed antibody supernatant incubated with the tissues (after pepsin and goat serum treatment) before washing and incubation with secondary antibodies. No staining was observed in tissues after this preabsorption (FigureS1 C). Specificity of the anti‐αSMA antibody used has been previously reported.4 Antibody specificity of the anti‐human IgG tagged with Dylight 650 (Abcam) was determined for the anti‐human IgG by staining sections from animals not been injected with FG‐3019.
Specificity of the CCN2 and collagen type I antibodies used for immuohistochemistry was determined using western blots to check if the antibodies detected bands at the correct molecular weights. Membranes were incubated with primary antibodies overnight at 4°C. For CCN2, gels were made as described earlier for CCN1 and 3. For collagen type I, 8% gels were made without SDS in the gel, yet with SDS in sample and loading buffers, as described.
Preabsorption antibody‐protein blocking steps were also performed to demonstrate either the CCN2 or collagen type 1 specificity using CCN2 recombinant protein (CXT‐687, Prospec, East Brunswick, NJ) and collagen type 1 rat protein (C7661, Sigma). A two‐ to threefold excess of purified protein, respectively, was preincubated with the matching antibody overnight at 4°C, the mixture centrifuged, and the preabsorbed antibody supernatant incubated with the tissues (after pepsin and goat serum treatment) before washing and incubation with secondary antibodies. No staining was observed in tissues after this preabsorption (Figure
Corresponding organizations : Temple University
3
Multiparametric Immunofluorescence Imaging of Tissue
For immunofluorescence (IF), paraffin-embedded tissue was dewaxed, and subjected to antigen retrieval by steaming samples in Target retrieval solution (pH 6.1) (DAKO, Carpinteria, CA, USA) or EDTA Decloaker (pH 8.0) (Biocare Medical, Concord, CA, USA) for at least 40 minutes before blocking with Protein Block (DAKO). The following primary antibodies were then used: CK8 (TROMA-1, 1:400, Developmental Studies Hybridoma Bank, Iowa City, IA, USA), α-SMA (A2547, 1:400, Sigma-Aldrich, St. Loius, MO, USA), PDGFR-α (3174, 1:100, Cell Signaling, Danvers, MA, USA), Ki67 (ab16667, 1:200, Abcam, Cambridge, UK), ERα (sc543, 1:10,000, Santa Cruz Biotechnology, Dallas, TX, USA) and CD31 (PECAM1- sc1506, 1:500, Santa Cruz). For IF, Secondary detection was performed using antibodies conjugated to AlexaFluor dyes (Invitrogen, Carlsbad, CA, USA) for 1 hour at room temperature. Sections were mounted with Prolong® Gold Antifade mount with DAPI (Life Technologies, Carlsbad, CA, USA). Representative fluorescent images were taken on an Eclipse E800 microscope (Nikon Instruments Inc., Melville, NY, USA) using the MetaVue™ Research Imaging system (Molecular Devices, Sunnyvale, CA, USA).
For immunohistochemistry, sections were stained using the Bond RX autostainer (Leica Biosystems Inc., Buffalo Grove, IL, USA). Slides were baked at 65 °C for 15 minutes and the automated system performed dewaxing, rehydration, antigen retrieval, blocking, primary antibody incubation, post primary antibody incubation, detection (DAB), and counterstaining using Bond reagents (Leica). Samples were then removed from the machine, dehydrated through a series of ethanol and xylenes and mounted. All quantitative imaging was done using the VECTRA® Automated Quantitative Pathology Imaging system (PerkinElmer, Waltham, MA, USA).
For immunohistochemistry, sections were stained using the Bond RX autostainer (Leica Biosystems Inc., Buffalo Grove, IL, USA). Slides were baked at 65 °C for 15 minutes and the automated system performed dewaxing, rehydration, antigen retrieval, blocking, primary antibody incubation, post primary antibody incubation, detection (DAB), and counterstaining using Bond reagents (Leica). Samples were then removed from the machine, dehydrated through a series of ethanol and xylenes and mounted. All quantitative imaging was done using the VECTRA® Automated Quantitative Pathology Imaging system (PerkinElmer, Waltham, MA, USA).
Corresponding organizations : The Ohio State University
4
Western Blot Analysis of Liver Proteins
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.
Corresponding organizations : Kagoshima University
5
Quantitative Western Blot Analysis
Western blot was conducted as we described previously27 (link) using the following primary antibodies: α-SMA, A2547, 1:1000 (Sigma); HDAC1, H3284, 1:1000 (Sigma); phophorylated SMAD3 (p-SMAD3), ab52903, 1:1000 (Abcam, Cambridge, MA). Protein band intensities were quantified using ImageJ software version 1.46 (National Institutes of Health).
Corresponding organizations : Saint Louis University
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