Abstract
For non-optically clear mammalian tissues, it is now possible to use multi-photon microscopy to penetrate deep into the tissue and obtain detailed single cell images in a live animal, i.e., intravital imaging. This technique is in principle applicable to any fluorescently marked cell, and we have employed it to observe stem cells during the regenerative process. Stem cell-mediated skeletal muscle regeneration in the mouse model has been classically studied at specific time points by sacrificing the animal and harvesting the muscle tissue for downstream analyses. A method for direct visualization of muscle stem cells to gain real-time information over a long period in a live mammal has been lacking. Here we describe a step-by-step protocol adapted from Webster et al. (2016) to quantitatively measure the behaviors of fluorescently labeled (GFP, EYFP) muscle stem and progenitor cells during homeostasis as well as following muscle injury.
Keywords: Muscle stem cell, Muscle progenitor, Muscle regeneration, Ghost fiber, Live imaging, Multi-photon microscopy, Second harmonic generation
Background
Long-term in vivo imaging of stem and progenitor cells was first used for hair follicles during continuous physiological regeneration without surgical procedure (Rompolas et al., 2012). By contrast, stem cells for skeletal muscles are largely quiescent and inactive during the normal homeostatic state. An injury to the muscle is necessary to activate muscle stem cells to mount a regenerative process. In vitro live imaging of muscle stem/progenitor cells has been widely used to study them in artificial settings. To understand muscle stem cell behavior during regeneration in their native environment, we developed a method to image them during skeletal muscle regeneration. Our method allows up to 8 h of continuous imaging per session daily following injury. This is the first time that skeletal muscle stem cells have been observed in vivo in an injured/regenerative environment (Webster et al., 2016).
Materials and Reagents
Equipment
Software
Procedure
Ethical statement: All procedures discussed here are in accordance with and were approved by the Carnegie Institution for Science Institutional Animal Care and Use Committee.
Data analysis
Data storage, transfer, and processing:
Notes
Acknowledgments
The protocol reported here was supported by the NIAMS of the NIH under award numbers F32AR065366 (to MTW) and RO1AR060042 (to C-MF), as well as by intramural funds of the Carnegie Institution (to TH). This protocol is a modified version derived from a previous publication in Cell Stem Cell (cited in the reference, Webster et al., 2016).
References
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