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Methods Mol Biol. 2016;1460:85-100. doi: 10.1007/978-1-4939-3810-0_8.

Isolation, Cryosection and Immunostaining of Skeletal Muscle.

Author information

1
Randall Division of Cell and Molecular Biophysics, King's College London, New Hunt's House, Guy's Campus, London, SE1 1UL, UK.
2
Department of Physiology, Graduate School of Health Sciences, Toyohashi SOZO University, 20-1 Matsushita, Ushikawa, Toyohashi, Aichi, 440-8511, Japan.
3
Randall Division of Cell and Molecular Biophysics, King's College London, New Hunt's House, Guy's Campus, London, SE1 1UL, UK. peter.zammit@kcl.ac.uk.

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.

KEYWORDS:

Cryosection; Immunostaining; Muscle fiber; Pax7; Satellite cell; Skeletal muscle; Stem cell

PMID:
27492168
DOI:
10.1007/978-1-4939-3810-0_8
[Indexed for MEDLINE]

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