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Isolation of Mouse Periocular Tissue for Histological and Immunostaining Analyses of the Extraocular Muscles and Their Satellite Cells.
Methods Mol Biol. 2016; 1460:101-27.MM

Abstract

The extraocular muscles (EOMs) comprise a group of highly specialized skeletal muscles controlling eye movements. Although a number of unique features of EOMs including their sparing in Duchenne muscular dystrophy have drawn a continuous interest, knowledge about these hard to reach muscles is still limited. The goal of this chapter is to provide detailed methods for the isolation and histological analysis of mouse EOMs. We first introduce in brief the basic anatomy and established nomenclature of the extraocular primary and accessory muscles. We then provide a detailed description with step-by-step images of our procedure for isolating (and subsequently cryosectioning) EOMs while preserving the integrity of their original structural organization. Next, we present several useful histological protocols frequently used by us, including: (1) a method for highlighting the general organization of periocular tissue, using the MyoD(Cre) × R26(mTmG) reporter mouse that elegantly distinguishes muscle (MyoD(Cre)-driven GFP(+)) from the non-myogenic constituents (Tomato(+)); (2) analysis by H&E staining, allowing for example, detection of the pathological features of the dystrophin-null phenotype in affected limb and diaphragm muscles that are absent in EOMs; (3) detection of the myogenic progenitors (i.e., satellite cells) in their native position underneath the myofiber basal lamina using Pax7/laminin double immunostaining. The EOM tissue harvesting procedure described here can also be adapted for isolating and studying satellite cells and other cell types. Overall, the methods described in this chapter should provide investigators the necessary tools for entering the EOM research field and contribute to a better understanding of this highly specialized muscle group and its complex micro-anatomy.

Authors+Show Affiliations

Department of Biological Structure, School of Medicine, University of Washington, Seattle, WA, USA.Department of Biological Structure, School of Medicine, University of Washington, Seattle, WA, USA. reuveni@uw.edu.

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

27492169

Citation

Stuelsatz, Pascal, and Zipora Yablonka-Reuveni. "Isolation of Mouse Periocular Tissue for Histological and Immunostaining Analyses of the Extraocular Muscles and Their Satellite Cells." Methods in Molecular Biology (Clifton, N.J.), vol. 1460, 2016, pp. 101-27.
Stuelsatz P, Yablonka-Reuveni Z. Isolation of Mouse Periocular Tissue for Histological and Immunostaining Analyses of the Extraocular Muscles and Their Satellite Cells. Methods Mol Biol. 2016;1460:101-27.
Stuelsatz, P., & Yablonka-Reuveni, Z. (2016). Isolation of Mouse Periocular Tissue for Histological and Immunostaining Analyses of the Extraocular Muscles and Their Satellite Cells. Methods in Molecular Biology (Clifton, N.J.), 1460, 101-27. https://doi.org/10.1007/978-1-4939-3810-0_9
Stuelsatz P, Yablonka-Reuveni Z. Isolation of Mouse Periocular Tissue for Histological and Immunostaining Analyses of the Extraocular Muscles and Their Satellite Cells. Methods Mol Biol. 2016;1460:101-27. PubMed PMID: 27492169.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Isolation of Mouse Periocular Tissue for Histological and Immunostaining Analyses of the Extraocular Muscles and Their Satellite Cells. AU - Stuelsatz,Pascal, AU - Yablonka-Reuveni,Zipora, PY - 2016/8/6/entrez PY - 2016/8/6/pubmed PY - 2018/1/13/medline KW - Antigen retrieval KW - Duchenne muscular dystrophy KW - Extraocular muscles KW - Laminin KW - MyoDCre × R26mTmG KW - Ocular/periocular tissue KW - Pax7 KW - Retractor bulbi muscle KW - Satellite cells KW - mdx4cv SP - 101 EP - 27 JF - Methods in molecular biology (Clifton, N.J.) JO - Methods Mol Biol VL - 1460 N2 - The extraocular muscles (EOMs) comprise a group of highly specialized skeletal muscles controlling eye movements. Although a number of unique features of EOMs including their sparing in Duchenne muscular dystrophy have drawn a continuous interest, knowledge about these hard to reach muscles is still limited. The goal of this chapter is to provide detailed methods for the isolation and histological analysis of mouse EOMs. We first introduce in brief the basic anatomy and established nomenclature of the extraocular primary and accessory muscles. We then provide a detailed description with step-by-step images of our procedure for isolating (and subsequently cryosectioning) EOMs while preserving the integrity of their original structural organization. Next, we present several useful histological protocols frequently used by us, including: (1) a method for highlighting the general organization of periocular tissue, using the MyoD(Cre) × R26(mTmG) reporter mouse that elegantly distinguishes muscle (MyoD(Cre)-driven GFP(+)) from the non-myogenic constituents (Tomato(+)); (2) analysis by H&E staining, allowing for example, detection of the pathological features of the dystrophin-null phenotype in affected limb and diaphragm muscles that are absent in EOMs; (3) detection of the myogenic progenitors (i.e., satellite cells) in their native position underneath the myofiber basal lamina using Pax7/laminin double immunostaining. The EOM tissue harvesting procedure described here can also be adapted for isolating and studying satellite cells and other cell types. Overall, the methods described in this chapter should provide investigators the necessary tools for entering the EOM research field and contribute to a better understanding of this highly specialized muscle group and its complex micro-anatomy. SN - 1940-6029 UR - https://www.unboundmedicine.com/medline/citation/27492169/Isolation_of_Mouse_Periocular_Tissue_for_Histological_and_Immunostaining_Analyses_of_the_Extraocular_Muscles_and_Their_Satellite_Cells_ L2 - https://dx.doi.org/10.1007/978-1-4939-3810-0_9 DB - PRIME DP - Unbound Medicine ER -