Mouse α actinin

Manufactured by Merck Group

Sourced in United States

Mouse α-actinin is a laboratory reagent that is a structural protein found in the cytoskeleton of eukaryotic cells. It functions in the organization and stabilization of actin filaments.

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7 protocols using mouse α actinin

Cryosectioning and Immunostaining of Mouse Embryos

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Mouse embryos were isolated in cold PBS and fixed in 4% PFA for 1~2 hr, followed by equilibration in 30% sucrose in PBS solution overnight. The tissues were placed in 1:1 30% sucrose/OCT (Tissue-Tek, Electron Microscopy Sciences) solution for 1–2 hr, then in 100% OCT compound for 1 hr at 4°C, and embedded in 100% OCT compound, carefully oriented in Cryomolds (Ted Pella). The blocks were immediately frozen on dry ice with isopropanol and stored at −80°C. The sections were cut 5 μm with a Leica CM3050 S cryostat. The following primary and secondary antibodies were used: α-actinin (mouse, 1:200, Sigma-Aldrich), Phospho-Histone 3 (rabbit, 1:250, Millipore), as well as Alexa Fluor 488 (green)- and Alexa Fluor 594 (red)-conjugated secondary antibodies specific to the appropriate species, which were used (1:500; ThermoFisher) for fluorescent staining. Sections were mounted with antifade mounting medium with DAPI (ThermoFisher), and analyzed by using AxioImager D1 (Carl Zeiss Microimaging, Inc).

Nakano H., Minami I., Braas D., Pappoe H., Wu X., Sagadevan A., Vergnes L., Fu K., Morselli M., Dunham C., Ding X., Stieg A.Z., Gimzewski J.K., Pellegrini M., Clark P.M., Reue K., Lusis A.J., Ribalet B., Kurdistani S.K., Christofk H., Nakatsuji N, & Nakano A. (2017). Glucose inhibits cardiac muscle maturation through nucleotide biosynthesis. eLife, 6, e29330.

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Spontaneous Differentiation of hiPSCs

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Human induced pluripotent stem cells were spontaneously differentiated in all three germlines by formation of embryoid bodies (EBs) in hESC medium (DMEM/F12 + GlutaMAX, 20% knockout serum replacement, 1% NEAA, 1% antibiotic-antimycotic [all Gibco), 1% β-mercaptoethanol (Millipore)] in low-attachment flasks. ROCK inhibitor was added the first 24 h. Cells were kept 7 days as free-floating EBs, plated on 35 mm μ-dishes (Ibidi) coated with poly-L-ornithine and laminin and grown further for 21 days. Cells were fixed and stained for ectoderm, mesoderm, and endoderm using the following primary antibodies: β-III-TUBULIN (chicken, 1:1,000, Millipore), α-Actinin (mouse, 1:500, Sigma-Aldrich) and alpha-fetoprotein (AFP, goat, 1:100, Santa-Cruz). hiPS cell lines were karyotyped to exclude cell lines-harboring chromosomal aberrations after reprogramming (Linta et al., 2012 (link)). hiPSCs were treated with 1.5 M Colchicine (Eurobio) diluted in mTeSR1 for 2 h at 37°C. Cells were trypsinized (TrypLE, Gibco), and metaphases were kindly analyzed by the Institute for Human Genetics, Ulm University.

Lutz A.K., Pérez Arévalo A., Ioannidis V., Stirmlinger N., Demestre M., Delorme R., Bourgeron T, & Boeckers T.M. (2021). SHANK2 Mutations Result in Dysregulation of the ERK1/2 Pathway in Human Induced Pluripotent Stem Cells-Derived Neurons and Shank2(−/−) Mice. Frontiers in Molecular Neuroscience, 14, 773571.

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Immunofluorescence Staining Protocol

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For immunofluorescence staining, cells were fixed with 4% formaldehyde, permeabilized with 0.1% triton X-100 in PBS, blocked with 3% FBS, incubated overnight with primary antibody (mouse α-actinin, Sigma, 7811) and troponin-I (rabbit, Santa Cruz Biotechnology, sc-15368) at 4°C diluted 1:400 in primary antibody, washed with PBS, followed by incubation with appropriate secondary antibody and counter-stained with DAPI.

Morales C.R., Li D.L., Pedrozo Z., May H.I., Jiang N., Kyrychenko V., Cho G., Kim S.Y., Wang Z.V., Rotter D., Rothermel B.A., Schneider J.W., Lavandero S., Gillette T.G, & Hill J.A. (2016). Inhibition of class I histone deacetylases blunts cardiac hypertrophy through TSC2-dependent mTOR repression. Science signaling, 9(422), ra34.

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Immunofluorescence Analysis of Rat Cardiomyocytes

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Cultured adult rat cardiomyocytes were plated on chamber slides (Lab-Tek II, Thermo Scientific) for immunofluorescence imaging. After 48 hours in culture, cells were rinsed twice with PBS and fixed with cold (4 °C) 4% paraformaldehyde for 3 minutes, followed by 1 minute in ice cold (−20 °C) methanol. Cells were then permeabilized in 0.5% Triton X-100 in PBS (20 minutes), 0.1% Triton X-100 twice (10 minutes), and antigen-retrieval solution (0.1 M glycine, pH 7.4) (30 minutes), followed by rinsing three times with PBS. Cells were blocked with 0.1% BSA, 0.1% gelatin, 0.1% Tween-20, and 0.0001% NaN₃, followed by incubation with primary antibodies for ssMyBP-C (ProSci 6679), fsMyBP-C (ProSci 5651), rabbit c-Myc (1:500 Roche) and mouse α-actinin (1:500 Sigma) overnight. After rinsing with PBS, corresponding secondary antibodies (AlexaFluor rabbit 488 at 1:50 dilution and mouse 568 at 1:50 dilution) were added for 1 hour at room temperature, rinsed, and coverslipped with VectaShield mounting medium (Vector Labs H-1500 10mUL 1.5ug/mL) for imaging with confocal microscopy. Images were taken using a Leica TCS SP5 and processed using ImageJ (NIH).

Lin B.L., Li A., Mun J.Y., Previs M.J., Previs S.B., Campbell S.G., dos Remedios C.G., Tombe P.D., Craig R., Warshaw D.M, & Sadayappan S. (2018). Skeletal myosin binding protein-C isoforms regulate thin filament activity in a Ca2+-dependent manner. Scientific Reports, 8, 2604.

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Immunostaining and Flow Cytometry of iPSC-CMs

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We applied 1:400 mouse α-Actinin (Sigma-Aldrich, St. Louis, MO, USA) for immunostaining. For flow cytometry, we applied 1:100 mouse Troponin-I (Ab19615, Abcam, Cambridge, UK) for the dissociated iPSC-CMs. (Full method can be found in the Supplementary Material).

Lee J.J., Cheng S.J., Huang C.Y., Chen C.Y., Feng L., Hwang D.Y., Kamp T.J., Chen H.C, & Hsieh P.C. (2019). Primary cardiac manifestation of autosomal dominant polycystic kidney disease revealed by patient induced pluripotent stem cell-derived cardiomyocytes. EBioMedicine, 40, 675-684.

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3D Reconstruction of Cardiac Embryos

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Embryos were fixed with 4% of paraformaldehyde overnight, dehydrated and embedded in paraffin, and cut at 8 µm slice thickness. One in four sections were used for immunohistochemistry to identify the myocardium by an overnight incubation at room temperature with mouse‐α‐actinin (Sigma‐Aldrich A9357, St. Louis, MO, US, diluted 1:400), followed by a 2‐hour incubation at room temperature with a goat‐anti‐mouse antibody conjugated with Alexa 488 (Thermo Fisher, Waltham, MA, US, diluted 1:250). DAPI was used for staining nuclei (Thermo Fisher, Waltham, MA, US, diluted 1:1000). Labelling α‐actinin‐positive cardiac tissue, calculating volume and generating 3D reconstructions were performed using Amira Software (version 5.3.3, Thermo Fisher Scientific, Waltham, MA, US) as described before.¹⁸ Cardiac nuclei count was determined using previously described custom software.⁵⁷

Marchal G.A., Verkerk A.O., Mohan R.A., Wolswinkel R., Boukens B.J, & Remme C.A. (2020). The sodium channel NaV1.5 impacts on early murine embryonic cardiac development, structure and function in a non‐electrogenic manner. Acta Physiologica (Oxford, England), 230(2), e13493.

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Cardiomyocyte contractility regulation

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M199 medium (Invitrogen UK, 11150), taurine (Biochemica, A1141), creatine monohydrate (Sigma Aldrich, C3630), penicillin/streptomycin (Merck, A2212), carnitine hydrochloride (Sigma Aldrich, C9500) BSA (Sigma Aldrich, A6003), laminin (Sigma Aldrich L2020), isoproterenol hydrochloride (Sigma Aldrich, I6504), ICI118551 (Tocris UK, 0821), CGP 20712A (Tocris UK, 1024), SR 59230A hydrochloride (Tocris UK, 1511), L-NAME hydrochloride (Tocris UK, 0665), Vinpocetine (Sigma Aldrich, V6383), EHNA hydrochloride (Sigma Aldrich, E114), Cilostamide (Tocris UK, 0915), Tadalafil (Santa Cruz USA, sc-208412), IBMX (Santa Cruz sc-201188), self-made rabbit sGCα and β subunit antibodies (specificity tested in KO animals; Friebe et al., 2018 (link)), mouse α-actinin (Sigma Aldrich, A7732), mouse Caveolin-3 (BD Transduction Laboratories, 610421, specificity tested in KO animals; Woodman et al., 2002 (link)), secondary Alexa Fluor antibodies 488 nm, 514 nm, 568 nm and 633 nm (Life Technologies), BSA (Fisher Scientific UK, BPE9704), fluorescence mounting medium (Vectashield Germany, H-1000), MaTek glass-bottom dishes (MaTek USA, P35G-1.5–10 C), TAT-scram and TAT-Cav3 peptides (a gift from Dr. Sarah Calaghan from Leeds, England).

Schobesberger S., Wright P.T., Poulet C., Sanchez Alonso Mardones J.L., Mansfield C., Friebe A., Harding S.E., Balligand J.L., Nikolaev V.O, & Gorelik J. (2020). β3-Adrenoceptor redistribution impairs NO/cGMP/PDE2 signalling in failing cardiomyocytes. eLife, 9, e52221.

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