Skeletal Muscle Tissue Clearing for LacZ and Fluorescent Reporters, and Immunofluorescence Staining
Skeletal muscle is a highly ordered yet complex tissue containing several cell types that interact with each other in order to maintain structure and homeostasis. It is also a highly regenerative tissue that responds to damage in a highly intricate but stereotypic manner, with distinct spatial and temporal kinetics. Proper examination of this process requires one to look at the three-dimensional orientation of the cellular and subcellular components, which can be accomplished through tissue clearing. While there has been a recent surge of protocols to study biology in whole tissue, it has primarily focused on the nervous system. This chapter describes the workflow for whole mount analysis of murine skeletal muscle for LacZ reporters, fluorescent reporters and immunofluorescence staining. Using this technique, we are able to visualize LacZ reporters more effectively in deep tissue samples, and to perform fluorescent imaging with a depth greater than 1700 μm.
Key wordsSkeletal muscle Tissue imaging tdTomato Green fluorescence protein (GFP) Satellite cell Tissue clearing lacZ reporter
- 7.Relaix F, Rocancourt D, Mansouri A, Buckingham M (2005) A Pax3/Pax7-dependent population of skeletal muscle progenitor cells. Nature 435:948–953. doi: 10.1038/nature03594
- 8.Asakura A, Seale P, Girgis-Gabardo A, Rudnicki MA (2002) Myogenic specification of side population cells in skeletal muscle. J Cell Biol 159:123–134. doi: 10.1083/jcb.200202092
- 9.Tajbakhsh S, Bober E, Babinet C et al (1996) Gene targeting the myf-5 locus with nlacZ reveals expression of this myogenic factor in mature skeletal muscle fibres as well as early embryonic muscle. Dev Dyn 206:291–300. doi: 10.1002/(SICI)1097-0177(199607)206:3<291::AID-AJA6>3.0.CO;2-D CrossRefPubMedGoogle Scholar