TY  - JOUR
TI  - An Integrated Workflow for Three-Dimensional Visualization of Human Skeletal Muscle Stem Cell Nuclei
AU  - Pearson, Jeremy R.
AU  - Martinez-Rivera, Noraida 
AU  - Torres-Vasquez, Irma 
AU  - Gallagher, Philip M.
AU  - Rosa-Molinar, Eduardo 
VL  - 15
IS  - 8
PY  - 2025
DA  - 2025/04/20
SP  - e5281
C1  - Bio-protocol 2025;15:e5281
DO  - 10.21769/BioProtoc.5281
UR  - https://doi.org/10.21769/BioProtoc.5281
AB  - Skeletal muscle–specific stem cells are responsible for regenerating damaged muscle tissue following strenuous physical activity. These muscle stem cells, also known as satellite cells (SCs), can activate, proliferate, and differentiate to form new skeletal muscle cells. SCs can be identified and visualized utilizing optical and electron microscopy techniques. However, studies identifying SCs using fluorescent imaging techniques vary significantly within their methodology and lack fundamental aspects of the guidelines for rigor and reproducibility that must be included within immunohistochemical studies. Therefore, a standardized method for identifying human skeletal muscle stem cells is warranted, which will improve the reproducibility of future studies investigating satellite activity. Additionally, although it has been suggested that SC shape can change after exercise, there are currently no methods for examining SC morphology. Thus, we present an integrated workflow for three-dimensional visualization of satellite cell nuclei, validated by the spatial context of the fluorescent labeling and multichannel signal overlap. Our protocol includes, from start to finish, post-biopsy extraction and embedding, tissue sectioning, immunofluorescence, imaging steps and acquisition, and three-dimensional data post-processing. Because of the depth volume generated from the confocal microscope z-stacks, this will allow future studies to investigate the morphology of SC nuclei and their activity, instead of traditionally observing them in two-dimensional space (x, y).
KW  - Satellite cells
KW  - Immunohistochemistry
KW  - Muscle regeneration
KW  - Fluorescent microscopy
KW  - Muscle damage
KW  - Myofiber cross-sectional area
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False