Alpha smooth muscle actin

Manufactured by Merck Group

Sourced in United Kingdom, United States

Alpha-smooth muscle actin is a protein component of the contractile apparatus in smooth muscle cells. It is a widely used marker for the identification and characterization of smooth muscle cells in various biological and medical applications.

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Lab products found in correlation

Calretinin, by Abcam (2 mentions) Flk 1, by BD (2 mentions) Texas red, by Jackson ImmunoResearch (2 mentions) Fluorescein isothiocyanate (fitc), by Jackson ImmunoResearch (2 mentions) Cd11b, by Santa Cruz Biotechnology (2 mentions) Pecam 1, by Santa Cruz Biotechnology (2 mentions) Vimentin, by Abcam (2 mentions) Acetylated tubulin, by Merck Group (2 mentions) Hoechst, by Thermo Fisher Scientific (2 mentions) Slowfade gold antifade reagent, by Thermo Fisher Scientific (2 mentions) Protein block, by Vector Laboratories (1 mentions) Foxa2, by Abcam (1 mentions) Epcam, by Abcam (1 mentions) Hnf4α, by Abcam (1 mentions) Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions) Bh 2 brightfield microscope, by Olympus (1 mentions) Tcs sp5 aobs confocal microscope system, by Zeiss (1 mentions) Axioscope m2, by Olympus (1 mentions) Elastin, by Abcam (1 mentions) Alpha smooth muscle actin, by Cell Signaling Technology (1 mentions)

15 protocols using alpha smooth muscle actin

Immunofluorescence Staining of Paraffin Sections

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Example 6

Paraffin sections were rehydrated through changes of xylene and graded alcohols. Slides were boiled in 10 mM citrate buffer with 0.05% Tween-20 at pH 6.0 for 20 min. Blocking was preformed with 3% BSA in PBS for one hour. Primary antibodies to PCNA, PECAM-1 (rabbit polyclonal, Santa Cruz), vWF, eNOS, Calretinin, Vimentin (rabbit polyclonal, Abcam), vWF (goat polyclonal, Santa Cruz), FLK-1 (mouse monoclonal, BD Bioscience), CD34, CD45, CD11b (mouse monoclonal, Santa Cruz), alpha-smooth muscle actin (mouse monoclonal, Sigma), and CD8 (rabbit monoclonal Abcam), were diluted to 10 μg/ml in PBS and incubated overnight at 4° C. Slides were washed with three changes of PBS with 0.05% Tween-20 between steps. Appropriate secondary antibodies conjugated to either FITC or Texas Red (Jackson Immunoresearch) were diluted 1:250 and incubated for one hour. The slides were mounted with DAPI-containing mounting medium and visualized on a Nikon Eclipse TE200 fluorescent microscope.

US11414644B2. Methods of recellularizing a tissue or organ for improved transplantability (2022-08-16). Regents of the University of Minnesota [US]. Inventors: Doris Taylor [US], Stefan M. Kren [US].

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Immunohistochemical Profiling of Liver Tissue

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Tissue was fixed in 10% buffered formalin for 8 h and embedded in paraffin for sectioning. Sections were dewaxed in xylene and rehydrated in decreasing concentrations of ethanol. Tissue underwent heat-induced antigen retrieval for 10 min in Tris-EDTA (pH = 9). For single chromogenic immunodetection, sections were blocked for endogenous peroxidase and avidin/biotin binding sites (Vector Laboratories, Burlingame, CA, USA). Primary antibodies were incubated overnight at 4 °C at the following concentrations: GFP (Abcam, 1:400), HNF4α (Abcam, 1:250), alpha-smooth muscle actin (ɑSMA; Sigma-Aldrich, 1:1000), Foxa2 (Abcam, Cambridge, UK, ab108422, 1:500), albumin (Abcam, 1:100), and epithelial cell adhesion molecule (EpCAM; Abcam, 1:100), and signal was visualized using avidin-biotin complex methods. For immunofluorescent detection, sections were blocked for an hour in protein block (Vector Laboratories) and primary antibodies were incubated overnight and visualized utilizing Alexa Fluor secondary antibodies (Invitrogen, 1:200) and DAPI (1:1000).

Orge I.D., Gadd V.L., Barouh J.L., Rossi E.A., Carvalho R.H., Smith I., Allahdadi K.J., Paredes B.D., Silva D.N., Damasceno P.K., Sampaio G.L., Forbes S.J., Soares M.B, & Souza B.S. (2020). Phenotype instability of hepatocyte-like cells produced by direct reprogramming of mesenchymal stromal cells. Stem Cell Research & Therapy, 11, 154.

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Temporal Dynamics of Hedgehog Signaling

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Immunohistochemical and fluorescence stains were performed on 5 μm, paraffin-embedded sections from E10.5, E13.5, E14.5, E15.5, E16.5, P0, and adult wildtype (6-month) and conditional Dhh mice as previously described (11 , 12 (link), 32 , 34 (link), 46 (link), 47 (link)). Primary antibodies and their dilutions included: acetylated tubulin (Sigma, #T6793, 1:500), alpha-smooth muscle actin (Sigma, #A2547, 1:500), alpha-smooth muscle actin (Cell Signaling, #19245, 1:500), ARL13B (Protein Tech, #17711–1-AP, 1:500), Collagen Telo (a generous gift from Dr. Stanley Hoffman, 1:250), DHH (Santa Cruz, #sc-271168, 1:50), Elastin (Abcam, #ab77804, 1:250), Smoothened (Novus, #NSL2666, 1:100), Versican (a generous gift from Dr. Stanley Hoffman, 1:250). Secondary antibodies were all purchased from Invitrogen, used at a 1:100 dilution, and included fluorophores 488, 568, and Cy5. Nuclei were stained with Hoechst (Life Technologies, #H3569, 1:10,000). Slides were cover-slipped using Invitrogen SlowFade Gold Antifade Reagent (#S36936). Images were captured using: Leica TCS SP5 AOBS Confocal Microscope System and LAS AF v2.6.3 Build 8173 Acquisition and Analysis Software, Zeiss Axioscope M2, or Olympus BH-2 brightfield microscope. n>3/genotype.

Fulmer D., Toomer K.A., Glover J., Guo L., Moore K., Moore R., Stairley R., Gensemer C., Abrol S., Rumph M.K., Emetu F., Lipschutz J., McDowell C., Bian J., Wang C., Beck T., Wessels A., Renault M.A, & Norris R. (2020). Desert Hedgehog-Primary Cilia Cross Talk Shapes Mitral Valve Tissue by Organizing Smooth Muscle Actin. Developmental biology, 463(1), 26-38.

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Oxidative Damage in Rat Lungs

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Rat lungs were collected and inflated with an 1% solution of low-melt-agarose and fixed in formalin and embedded in paraffin. Five micrometer-thick lung paraffin sections were deparaffinized and rehydrated. Sections were boiled for 40 min in Vector^® Antigen Unmasking Solution (Vector, Burlingame, USA) using a pressure cooker. After blocking with goat serum 10% (ThermoFisher Scientific, Massachusetts, USA), sections were incubated overnight at 4 °C with primary antibodies directed against 8-OHdG (1:150; bs-1278R, Bioss Antibodies, Woburn, MA, USA), co-stained with von Willebrand factor (1:1000; ab8822, Abcam, Cambridge, UK), and alpha smooth muscle actin (1:1000; Sigma-Aldrich, St. Louis, MO, USA). All sections were mounted with ProLong^® Gold antifade reagent (Invitrogen, Massachusetts, USA) containing DAPI.

Gomez-Puerto M.C., Sun X.Q., Schalij I., Orriols M., Pan X., Szulcek R., Goumans M.J., Bogaard H.J., Zhou Q, & ten Dijke P. (2020). MnTBAP Reverses Pulmonary Vascular Remodeling and Improves Cardiac Function in Experimentally Induced Pulmonary Arterial Hypertension. International Journal of Molecular Sciences, 21(11), 4130.

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Characterization of HUVEC Differentiation under MTX

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To characterize differentiation of HUVECs while MTX addition, the expression of extra cellular matrix proteins (e.g. CD31, von Willebrand factor, alpha smooth muscle actin, SigmaAldrich Co., St. Louis, MO, USA) was measured. Additional culture dishes (0-1000 nM MTX) were used for immunostaining. When control samples reached confluence, the expression of CD 31 and von Willebrand factor (vWF) as well as alpha smooth muscle actin (α-SMA, negative control) was determined. Cells were washed twice with phosphate buffered saline and fixated for 20 minutes at −20°C. One hundred microL of blocking solution (CANDOR Bioscience, Wangen, Germany) was used for each sample and a period of 15 minutes. According to the manufacture’s instruction, primary antibodies (CD 31, vWF, α-SMA) as well as secondary antibodies (Alexa Fluor 488, Life Technologies, Carlsbad, Canada) were incubated for 60 minutes at 37°C (n = 30).

Annussek T., Szuwart T., Kleinheinz J., Koiky C, & Wermker K. (2014). In vitro inhibition of HUVECs by low dose methotrexate – insights into oral adverse events. Head & Face Medicine, 10, 19.

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Immunophenotyping of Stem Cell Cultures

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ADEPCs, ADSCs and RBSMCs were cultured on glass slides in a 24-well plate pre-coated with 0.1% gelatin for facilitating cell adhesion. Immunofluorescent staining with primary membranous (CD31, CD34, Abcam) and cytoplasmic (stromal cell antigen [Stro-1], Millipore; endothelial nitric oxide synthase [eNOS], BD Biosciences; alpha-smooth muscle actin [α-SMA], Sigma-Aldrich) antibodies was done. An IgG-matched isotype served as the internal control for each antibody.

Zhou L., Xia J., Qiu X., Wang P., Jia R., Chen Y., Yang B, & Dai Y. (2015). In Vitro Evaluation of Endothelial Progenitor Cells from Adipose Tissue as Potential Angiogenic Cell Sources for Bladder Angiogenesis. PLoS ONE, 10(2), e0117644.

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Pluripotency and Lineage Marker Detection

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Cells were fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.5% Tween-20 in PBS, and incubated to 0.1% Tween-20 with 10% horse serum. We exposed the cells to primary antibodies overnight and to secondary antibodies for 1 hour (Alexa Fluor, Invitrogen). We employed the following primary antibodies: SSEA-3 (1:100, R&D), SSEA-4 (1:500, DSHB), TRA1-60 (1:500, Chemicon), TRA1-81 (1:500, Chemicon), NANOG (1:500 Abcam), alpha-fetoprotein (1:500, DAKO), alpha-actinin (1:500 Sigma), alpha smooth muscle actin (1:500, Sigma), Desmin (1:100, Novocastra), TuJ1 (1:1000, Sigma), and glial fibrillary acidic protein (GFAP, 1:1000, DAKO; 1:500, Sigma). Alkaline phosphatase staining was performed20 (link). For the analysis, we used a confocal LEICA LCS2 microscope.

Nizzardo M., Simone C., Dametti S., Salani S., Ulzi G., Pagliarani S., Rizzo F., Frattini E., Pagani F., Bresolin N., Comi G, & Corti S. (2015). Spinal muscular atrophy phenotype is ameliorated in human motor neurons by SMN increase via different novel RNA therapeutic approaches. Scientific Reports, 5, 11746.

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Immunofluorescence Staining of cVICs

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cVICs were seeded into 4-well, collagen-coated chamber slides and cultured with normal media at 37°C for 16 hours. Cells were serum starved for 5 hours and treated in serum free media for 2 hours. Media was aspirated and cells were fixed in 4% paraformaldehyde (PFA) for 15 minutes and permeabilized in 0.1% Triton-X for 5 minutes. Fixed cells were washed twice in PBS and blocked with 1% bovine serum albumin (BSA) in PBS for 1 hour at room temperature. Cells were incubated in primary antibody diluted in 1% BSA for 16 hours at 4°C or 1.5 hours at room temperature as necessary, washed twice with PBS and incubated in secondary antibody for 1 hour at room temperature. Slides were washed three times for 5 minutes in PBS, including a wash step with Hoechst (Life Technologies, #H3569, 1:10,000). Slides were coverslipped with Invitrogen SlowFade Gold Antifade Reagent (#S36936). Primary antibodies and their dilutions included: acetylated tubulin (Sigma, #T6793, 1:500), alpha-smooth muscle actin (Sigma, #A2547, 1:500), Collagen Telo (a generous gift from Dr. Stanley Hoffman, 1:250). Secondary antibodies were purchased from Invitrogen, used at a 1:100 dilution, and included fluorophores 488, 568, and Cy5. n=4 slide chambers/treatment group.

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Immunofluorescence Analysis of YAP/WWTR1 and Cytoskeletal Markers

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Cells from culture were fixed in 4% PFA for 15 minutes at room temperature. Cryosections from sham, incision control and STC or BSMC were fixed in 4% PFA for 20 minutes at RT, then stopped in 0.1 M Glycine in PBS. After washing, the samples were then incubated with 0.2% Triton X-100 for 10 minutes at RT, and subsequently washed 3 times in PBS. Nonspecific sites were blocked with 10% Donkey Serum + 10% Bovine Serum Albumin for 1 hour, room temperature. Primary antibodies (YAP/WWTR1, 1:100—Santa Cruz Biotechnology, Inc., Dallas, Texas, USA, beta-3 tubulin, 1:3000—abcam, calponin and alpha-smooth muscle actin, 1:200—Sigma) or normal IgG dilution in block, were incubated on sections or cells at 4°C overnight, followed by 3 washes in PBS. Secondary antibodies (1:1000 dilution in block) were incubated 1 hour at room temperature. Samples were then incubated with nuclear staining dye (Hoechst), washed with 0.1% Triton X-100 in PBS and mounted in Dako Fluorescence mounting medium. For YAP/WWTR1, methanol was used to pre-clear samples prior to the blocking step [26 , 33 ]. Fluorescence was imaged using spinning disk confocal microscopy and intensity of signals measured using Volocity software 6.3.

Aitken K.J., Yadav P., Sidler M., Thanabalasingam T., Ahmed T., Aggarwal P., Yip S.T., Jeffrey N., Jiang J.X., Siebenaller A., Sotiropoulos C., Huang R., Le D.M., Delgado-Olguin P, & Bagli D. (2023). Spontaneous urinary bladder regeneration after subtotal cystectomy increases YAP/WWTR1 signaling and downstream BDNF expression: Implications for smooth muscle injury responses. PLOS ONE, 18(7), e0287205.

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Histological Analysis of Kidney Fibrosis

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Histological analysis of fibrosis was performed on fixed renal tissue, embedded in paraffin, and sectioned at a thickness of 4 μm. Connective tissue deposition was examined with Picrosirius Red Stain Kit (Polysciences, Inc, Warrington, PA, USA) and collagen I (1:100, Millipore, Billerica, MA, USA), staining, as we have previously described [20 (link)–22 (link)]. Additionally, co-staining of CoRL and a myofibroblast marker alpha smooth muscle actin (αSMA, 1:10.000; Sigma, Saint Louis, MI, USA) were performed to determine whether CoRL become myofibroblasts in kidney fibrosis.

Stefanska A., Eng D., Kaverina N., Pippin J.W., Gross K.W., Duffield J.S, & Shankland S.J. (2016). Cells of renin lineage express hypoxia inducible factor 2α following experimental ureteral obstruction. BMC Nephrology, 17, 5.

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