Abstract
Adult skeletal muscle is maintained and repaired by resident stem cells called satellite cells, located between the plasmalemma of a muscle fiber, and the surrounding basal lamina. When needed, satellite cells are activated to form proliferative myoblasts, that then differentiate and fuse to existing muscle fibers, or fuse together to form replacement myofibers. In parallel, a proportion of satellite cells self-renew, to maintain the stem cell pool. To date, Pax7 is the marker of choice for identifying quiescent satellite cells. Co-immunostaining of skeletal muscle with Pax7 and laminin allows both identification of satellite cells, and the myofiber that they are associated with. Furthermore, satellite cells can be followed through the early stages of the myogenic program by co-immunostaining with myogenic regulatory factors such as MyoD. To test genetically modified mice for satellite cell expression, co-immunostaining can be performed for Pax7 and reporter genes such as eGFP. Here, we describe a method for identification of satellite cells in skeletal muscle sections, including muscle isolation, cryosectioning and co-immunostaining for Pax7 and laminin.
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References
Janssen I, Heymsfield SB, Wang ZM, Ross R (2000) Skeletal muscle mass and distribution in 468 men and women aged 18–88 yr. J Appl Physiol 89:81–88
Scharner J, Zammit PS (2011) The muscle satellite cell at 50: the formative years. Skelet Muscle 1:28
Mauro A (1961) Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol 9:493–495
Katz B (1961) The terminations of the afferent nerve fibre in the muscle spindle of the frog. Philos Trans R Soc Lond (Biol) 243:221–240
Schmalbruch H, Lewis DM (2000) Dynamics of nuclei of muscle fibers and connective tissue cells in normal and denervated rat muscles. Muscle Nerve 23:617–626
Schultz E, Gibson MC, Champion T (1978) Satellite cells are mitotically quiescent in mature mouse muscle: an EM and radioautographic study. J Exp Zool 206:451–456
Relaix F, Zammit PS (2012) Satellite cells are essential for skeletal muscle regeneration: the cell on the edge returns centre stage. Development 139:2845–2856
Negroni E, Vallese D, Vilquin JT, Butler-Browne G, Mouly V, Trollet C (2011) Current advances in cell therapy strategies for muscular dystrophies. Expert Opin Biol Ther 11:157–176
Seale P, Sabourin LA, Girgis-Gabardo A, Mansouri A, Gruss P, Rudnicki MA (2000) Pax7 is required for the specification of myogenic satellite cells. Cell 102:777–786
Gunther S, Kim J, Kostin S, Lepper C, Fan CM, Braun T (2013) Myf5-positive satellite cells contribute to Pax7-dependent long-term maintenance of adult muscle stem cells. Cell Stem Cell 13:590–601
Buckingham M, Relaix F (2015) PAX3 and PAX7 as upstream regulators of myogenesis. Semin Cell Dev Biol 44:115–25
Gnocchi VF, White RB, Ono Y, Ellis JA, Zammit PS (2009) Further characterisation of the molecular signature of quiescent and activated mouse muscle satellite cells. PLoS One 4:e5205
Halevy O, Piestun Y, Allouh MZ, Rosser BW, Rinkevich Y, Reshef R, Rozenboim I, Wleklinski-Lee M, Yablonka-Reuveni Z (2004) Pattern of Pax7 expression during myogenesis in the posthatch chicken establishes a model for satellite cell differentiation and renewal. Dev Dyn 231:489–502
Morrison JI, Loof S, He P, Simon A (2006) Salamander limb regeneration involves the activation of a multipotent skeletal muscle satellite cell population. J Cell Biol 172:433–440
Zammit PS, Partridge TA, Yablonka-Reuveni Z (2006) The skeletal muscle satellite cell: the stem cell that came in from the cold. J Histochem Cytochem 54:1177–1191
Zammit PS, Golding JP, Nagata Y, Hudon V, Partridge TA, Beauchamp JR (2004) Muscle satellite cells adopt divergent fates: a mechanism for self-renewal? J Cell Biol 166:347–357
Collins CA, Olsen I, Zammit PS, Heslop L, Petrie A, Partridge TA, Morgan JE (2005) Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell 122:289–301
Zammit PS, Heslop L, Hudon V, Rosenblatt JD, Tajbakhsh S, Buckingham ME, Beauchamp JR, Partridge TA (2002) Kinetics of myoblast proliferation show that resident satellite cells are competent to fully regenerate skeletal muscle fibers. Exp Cell Res 281:39–49
Rosenblatt JD, Parry DJ (1992) Gamma irradiation prevents compensatory hypertrophy of overloaded mouse extensor digitorum longus muscle. J Appl Physiol 73:2538–2543
van der Loos CM (2007) A focus on fixation. Biotech Histochem 82:141–154
Taylor CR, Shi SR, Chen C, Young L, Yang C, Cote RJ (1996) Comparative study of antigen retrieval heating methods: microwave, microwave and pressure cooker, autoclave, and steamer. Biotech Histochem 71:263–270
Shi SR, Key ME, Kalra KL (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 39:741–748
van Essen HF, Verdaasdonk MA, Elshof SM, de Weger RA, van Diest PJ (2010) Alcohol based tissue fixation as an alternative for formaldehyde: influence on immunohistochemistry. J Clin Pathol 63:1090–1094
Collins CA, Zammit PS (2009) Isolation and grafting of single muscle fibres. Methods Mol Biol 482:319–330
Moyle LA, Zammit PS (2014) Isolation, culture and immunostaining of skeletal muscle fibres to study myogenic progression in satellite cells. Methods Mol Biol 1210:63–78
Fortier M, Figeac N, White RB, Knopp P, Zammit PS (2013) Sphingosine-1-phosphate receptor 3 influences cell cycle progression in muscle satellite cells. Dev Biol 382:504–516
Acknowledgements
We would like to thank members of the Zammit and Goto labs for help and advice. We also gratefully acknowledge colleagues who shared antibodies through the Developmental Studies Hybridoma Bank developed under the Auspices of the NICHD and maintained by the University of Iowa. The laboratory of Professor Peter Zammit is supported by the Medical Research Council, Muscular Dystrophy UK, Association Française contre les Myopathies, FSHSoc and Rational Bioactive Materials Design for Tissue Regeneration (BIODESIGN) (262948 from the European Commission Seventh Framework Program.
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Ortuste Quiroga, H.P., Goto, K., Zammit, P.S. (2016). Isolation, Cryosection and Immunostaining of Skeletal Muscle. In: Kyba, M. (eds) Skeletal Muscle Regeneration in the Mouse. Methods in Molecular Biology, vol 1460. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-3810-0_8
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DOI: https://doi.org/10.1007/978-1-4939-3810-0_8
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