Anti sarcomeric alpha actinin

Manufactured by Abcam

Sourced in Germany, United States

Anti-sarcomeric alpha actinin is a protein that plays a crucial role in the structure and function of muscle cells. It is a key component of the sarcomere, the basic contractile unit of muscle fibers. Anti-sarcomeric alpha actinin is used in research to study the organization and dynamics of the cytoskeleton in muscle cells.

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Lab products found in correlation

Anti phospho histone h3 ser10, by Merck Group (2 mentions) Picric acid solution, by Merck Group (1 mentions) Nanoorange protein quantitation kit, by Thermo Fisher Scientific (1 mentions) Direct red 80, by Merck Group (1 mentions) Anti alpha smooth muscle actin, by Abcam (1 mentions) Recombinant human tgf β1, by Thermo Fisher Scientific (1 mentions) Fast green fcf, by Merck Group (1 mentions) Sodium hyaluronate, by Lifecore Biomedical (1 mentions) Triethylamine, by Merck Group (1 mentions) Dimethylformamide, by Merck Group (1 mentions) Glycidyl methacrylate, by Merck Group (1 mentions) Protein lobind microcentrifuge tubes, by Eppendorf (1 mentions) Axiovert 200 fluorescence microscope, by Zeiss (1 mentions) Prolong gold antifade mount with dapi, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 568 conjugated secondary antibodies, by Thermo Fisher Scientific (1 mentions) Axiovision 4, by Zeiss (1 mentions) Anti cardiac troponin t, by Abcam (1 mentions) Triton x 100, by Merck Group (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Methanol, by Carl Roth (1 mentions)

6 protocols using anti sarcomeric alpha actinin

Chondrocyte Differentiation Protocol

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Sodium hyaluronate (100kDa) was obtained from Lifecore Biomedical and stored at −20°C until use. Dimethylformamide (DMF), glycidyl methacrylate (GM), triethylamine (TEA), 2-hydroxy-4’-(2-hydroxyethoxy)-2-methylpropiophenone (#410896), differentiation solution (A3179-1L), 2-methylbutane, picric acid solution (P6744), Fast Green FCF (F7252), and Direct Red 80 (#365548) were purchased from Sigma-Aldrich (St. Louis, MO) and used as received. Spectrum^™ Spectra/Por^™ 3 RC dialysis membrane tubing, 3500 Dalton MWCO (#08-670-5B), Wheat Germ Agglutinin (WGA) Alexa Fluor 488 (#W11261), Hoescht (#33342), high concentration rat tail collagen type I (#CB354249), and NanoOrange Protein Quantitation Kit (#N6666) were purchased from Thermo Fisher Scientific (Waltham, MA). Protein LoBind microcentrifuge tubes (#022431102) were purchased from Eppendorf (Hamburg, Germany). OCT was obtained from Sakura Finetech USA, Inc. (Torrance, CA). Harris Hematoxylin Stain (#95057-858) and Eosin Y Solution (#95057-848) were purchased from VWR (Radnor, PA). Recombinant human TGF-β1 (#100 - 21) was obtained from PeproTech. Recombinant human decorin protein (ab167743), human decorin ELISA kit (ab99998), anti-decorin antibody (ab151988), anti-sarcomeric alpha actinin (ab137346), and anti-alpha smooth muscle actin (ab5694) were purchased from Abcam (Cambridge, UK).

Desai T., Mohindra P., Zhong J., Fang Q., Huynh C., Cuylear D., Qiu H., Gao D., Kharbikar B., Huang X., Springer M, & Lee R. (2023). Local delivery of decorin via hyaluronic acid microrods improves cardiac performance and ventricular remodeling after myocardial infarction. Research Square.

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Immunocytochemistry Analysis of iPSC-CMs

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For immunocytochemistry analysis, control, doxorubicin-exposed and washout iPSC-CMs were fixed with ice-cold 99 % methanol (Roth, Karlsruhe, Germany) for 10 min at −20 °C. Then, cells were permeabilized with 0.3 % Triton X-100 (Sigma-Aldrich, Steinheim, Germany) for 20 min at room temperature. Cells were blocked with 5 % bovine serum albumin (Sigma, Steinheim, Germany) for 1 h at room temperature and incubated with anti-sarcomeric alpha actinin (Abcam, 1:200) and anti-cardiac troponin T (Abcam, 1:200) for 1 h at 37 °C. The cells were washed 3 times with phosphate-buffered saline (PBS) with Ca²⁺ and Mg²⁺ for 5 min. Primary antibodies were detected using species matched respective Alexa Fluor-488/568-conjugated secondary antibodies (Invitrogen, Darmstadt, Germany) with 1 h incubation at 37 °C. The cells were washed 3 times with PBS for 5 min and then mounted with Prolong® Gold anti-fade mount with DAPI (Invitrogen, Darmstadt, Germany). Cell images were taken with an Axiovert 200 fluorescence microscope and Axiovision 4.3 software (Carl Zeiss).

Chaudhari U., Nemade H., Wagh V., Gaspar J.A., Ellis J.K., Srinivasan S.P., Spitkovski D., Nguemo F., Louisse J., Bremer S., Hescheler J., Keun H.C., Hengstler J.G, & Sachinidis A. (2015). Identification of genomic biomarkers for anthracycline-induced cardiotoxicity in human iPSC-derived cardiomyocytes: an in vitro repeated exposure toxicity approach for safety assessment. Archives of Toxicology, 90(11), 2763-2777.

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Multiparametric Immunohistochemistry: Cellular Analysis

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Following antigen retrieval with 1mM EDTA in boiling water, sections were blocked with 10% serum from host animal of secondary antibodies, and incubated with primary antibodies overnight at 4°C. Sections were subsequently washed with PBS and incubated with corresponding secondary antibodies conjugated to Alexa Fluor 350, 488 or 555 (Invitrogen). Primary antibodies used are following: anti-phospho Histone H3 Ser10 (Millipore #06–570, 1:100), anti-Aurora B (Sigma A5102, 1:100), anti-Troponin T, Cardiac Isoform Ab-1, Clone 13–11 (Thermo scientific MS-295-P1, 1:100), anti-Bromodeoxyuridine (Roche #11170376001, 1:25), anti-Sarcomeric Alpha Actinin (Abcam, ab68167, 1:100), anti-oxoguanine 8 (Abcam Ab64548, 1:100), anti-phosphorilated ATM (Santa Cruz Biotechnology sc-47739, 1:100), anti-Wee1 (Abcam ab137377, 1:100). Anti-weat germ agglutinin (WGA) conjugated to Alexa Fluor 488 (50 µg/ml, Invitrogen).

Puente B.N., Kimura W., Muralidhar S.A., Moon J., Amatruda J.F., Phelps K.L., Grinsfelder D., Rothermel B.A., Chen R., Garcia J.A., Santos C.X., Thet S., Mori E., Kinter M.T., Rindler P.M., Zacchigna S., Mukherjee S., Chen D.J., Mahmoud A.I., Giacca M., Rabinovitch P.S., Aroumougame A., Shah A.M., Szweda L.I, & Sadek H.A. (2014). The Oxygen Rich Postnatal Environment Induces Cardiomyocyte Cell Cycle Arrest Through DNA Damage Response. Cell, 157(3), 565-579.

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Immunostaining of Cardiac Muscle Cells

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Staining was performed as described^{28 (link),55 (link),56 (link)}. Primary antibodies: mouse monoclonal anti-mCherry (1:200, Clontech, 632543 or 1:50, DSHB, 3A11), anti-Tropomyosin (1:200, Sigma, T9283), anti-sarcomeric alpha Actinin (1:100, Abcam, ab9465), anti-Aurora B (1:200, BD Transduction Laboratories, 611083), anti-p27 (1:50, BD Transduction Laboratories, 610242), anti-Ki67 (1:250, Abcam, ab8191), rabbit polyclonal anti-Troponin I (sc-15368), anti-Cyclin A (sc-751), anti-cdc2 (sc-954), anti-Geminin (sc-13015) (all 1:50, Santa Cruz), anti-phospho-Histone H3 (Ser10) (1:200, Millipore, 06-570), anti-pRb807/811 (1:100, Cell Signaling, 9308), anti-mAG (1:300, MBL, PM011), anti p27 (1:50, SantaCruz, sc-528), anti-survivin (1:50, Novus, NB500-201K8), rat monoclonal anti-BrdU (1:100, Abcam, ab6326) and goat polyclonal anti c-kit (1:100, R&D Systems, AF1356). Immune complexes were detected with ALEXA 488- or ALEXA 594-conjugated secondary antibodies (1:200; Molecular Probes). DNA was visualized with DAPI (4′, 6′-diamidino-2-phenylindole, 0.5 g/ml). For BrdU, cells were cultured in 30 μM BrdU (neonatal: last 24 h, adult: last 5 days).

Magadum A., Ding Y., He L., Kim T., Vasudevarao M.D., Long Q., Yang K., Wickramasinghe N., Renikunta H.V., Dubois N., Weidinger G., Yang Q, & Engel F.B. (2017). Live cell screening platform identifies PPARδ as a regulator of cardiomyocyte proliferation and cardiac repair. Cell Research, 27(8), 1002-1019.

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Immunostaining of NLRP3, ASC, and Caspase-1 in H/R-stressed Cells

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CFs cultured alone or in the presence of CMs were cultured on glass coverslips in 6-well plates. The cells were subjected to H/R as described above. Then, the cells were incubated with 4% fixative solution (Solarbio, China) for 30 min and blocked in 10% normal goat serum (Solarbio, China)/PBS for 30 min. The cells were incubated overnight at 4 °C with anti-NLRP3 (1:200), anti-ASC (1:200), anti-cleaved-caspase1 (1:200), anti-sarcomeric alpha actinin (1:200, Abcam, USA) and anti-Vimentin (1:200, Abcam, USA) primary antibodies. Thereafter, the cells were washed with PBS and incubated with goat anti-rabbit IgG/FITC (1:500, MultiSciences, China), goat anti-chicken IgG/Cy5 (1:500, Solarbio, China), and Cy3-conjugated Affinipure goat anti-mouse IgG (1:50, Proteintech, USA) secondary antibodies (30 min, room temperature) to visualize NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), cleaved caspase1, Vimentin and sarcomeric alpha actinin. We used 4′,6-diamidino-2-phenylindole (DAPI) (1:200, Beyotime, China) to visualize the nuclei. Finally, the fluorescence was visualized using a fluorescence microscope. Three fields of view were randomly selected to observe the fluorescent protein in each batch of cells by one blinded for allocation.

Huang Y., Sun X., Juan Z., Zhang R., Wang R., Meng S., Zhou J., Li Y., Xu K, & Xie K. (2021). Dexmedetomidine attenuates myocardial ischemia-reperfusion injury in vitro by inhibiting NLRP3 Inflammasome activation. BMC Anesthesiology, 21, 104.

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Multiparametric Immunoprofiling of Muscle Differentiation

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Constructs were fixed with 2.5% paraformaldehyde for 30 min. Permeabilization and blocking steps were performed with Triton X-100 (0.1%) and Bovine Serum Albumin (5%). Cells were then probed with the following primary antibodies (overnight at 4 C): anti-a Smooth Muscle Actin (1:60, mouse anti-human, Abcam, distributed in Italy by Prodotti Gianni) to highlight differentiation of MSCs into mural cells; anti-Fast Myosin Heavy Chain (1:100, mouse anti-human, Abcam), anti-sarcomeric alpha actinin (1:100, mouse anti-human, Abcam) and anti-dystrophin (1:100, rabbit anti-human, Abcam) to highlight muscle cells differentiation; anti-Pax7 (1:100, mouse anti-human, Santa Cruz, distributed in Italy by DBA) for satellite cells; anti-collagen type I (1:250, rabbit anti-human, Abcam) and anti-fibronectin (1:200, rabbit anti-human, Abcam) for ECM protein deposition; anti-TSPAN7 (1:25, rabbit anti-human, Santacruz) for EC differentiation. Samples were washed with Phosphate Buffered Saline (PBS) and overnight incubated with secondary antibodies at 4 C (Alexa Fluor 488 or Alexa Fluor 568 or Alexa Fluor 647). Vybrant cell labeling (DiD, LifeTech) was employed to track muscle-derived fibroblasts. Nuclear staining was performed with DAPI whereas actin fibers staining was performed with Phalloidin.

Bersini S., Gilardi M., Ugolini G.S., Sansoni V., Talò G., Perego S., Zanotti S., Ostano P., Mora M., Soncini M., Vanoni M., Lombardi G, & Moretti M. (2018). Engineering an Environment for the Study of Fibrosis: A 3D Human Muscle Model with Endothelium Specificity and Endomysium. Cell reports, 25(13).

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