An Integrated Workflow for Three-Dimensional Visualization of Human Skeletal Muscle Stem Cell Nuclei
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 vi...
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Bio-protocol LLC
2025-04-01
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| Series: | Bio-Protocol |
| Online Access: | https://bio-protocol.org/en/bpdetail?id=5281&type=0 |
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| author | Jeremy Pearson Noraida Martinez-Rivera Irma Torres-Vasquez Philip Gallagher Eduardo Rosa-Molinar |
| author_facet | Jeremy Pearson Noraida Martinez-Rivera Irma Torres-Vasquez Philip Gallagher Eduardo Rosa-Molinar |
| author_sort | Jeremy Pearson |
| collection | DOAJ |
| description | 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). |
| format | Article |
| id | doaj-art-f2444ee301e44561ba8a85b68c0c04d8 |
| institution | DOAJ |
| issn | 2331-8325 |
| language | English |
| publishDate | 2025-04-01 |
| publisher | Bio-protocol LLC |
| record_format | Article |
| series | Bio-Protocol |
| spelling | doaj-art-f2444ee301e44561ba8a85b68c0c04d82025-08-20T03:11:21ZengBio-protocol LLCBio-Protocol2331-83252025-04-0115810.21769/BioProtoc.5281An Integrated Workflow for Three-Dimensional Visualization of Human Skeletal Muscle Stem Cell NucleiJeremy Pearson0Noraida Martinez-Rivera1Irma Torres-Vasquez2Philip Gallagher3Eduardo Rosa-Molinar4Microscopy and Analytical Imaging Research Resource Core Laboratory, University of Kansas, Lawrence, KS, USAApplied Physiology Laboratory and Osness Human Performance Laboratories, University of Kansas, Lawrence, KS, USAMicroscopy and Analytical Imaging Research Resource Core Laboratory, University of Kansas, Lawrence, KS, USADepartment of Pharmacology & Toxicology, University of Kansas, Lawrence, KS, USAApplied Physiology Laboratory and Osness Human Performance Laboratories, University of Kansas, Lawrence, KS, USAMicroscopy and Analytical Imaging Research Resource Core Laboratory, University of Kansas, Lawrence, KS, USADepartment of Pharmacology & Toxicology, University of Kansas, Lawrence, KS, USA, Department of Cell Biology & Physiology, and Neuroscience, Washington University Center for Cellular Imaging, Washington University School of Medicine, St Louis, MO, USASkeletal 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).https://bio-protocol.org/en/bpdetail?id=5281&type=0 |
| spellingShingle | Jeremy Pearson Noraida Martinez-Rivera Irma Torres-Vasquez Philip Gallagher Eduardo Rosa-Molinar An Integrated Workflow for Three-Dimensional Visualization of Human Skeletal Muscle Stem Cell Nuclei Bio-Protocol |
| title | An Integrated Workflow for Three-Dimensional Visualization of Human Skeletal Muscle Stem Cell Nuclei |
| title_full | An Integrated Workflow for Three-Dimensional Visualization of Human Skeletal Muscle Stem Cell Nuclei |
| title_fullStr | An Integrated Workflow for Three-Dimensional Visualization of Human Skeletal Muscle Stem Cell Nuclei |
| title_full_unstemmed | An Integrated Workflow for Three-Dimensional Visualization of Human Skeletal Muscle Stem Cell Nuclei |
| title_short | An Integrated Workflow for Three-Dimensional Visualization of Human Skeletal Muscle Stem Cell Nuclei |
| title_sort | integrated workflow for three dimensional visualization of human skeletal muscle stem cell nuclei |
| url | https://bio-protocol.org/en/bpdetail?id=5281&type=0 |
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