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 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). Key features • Detailed instructions on post-biopsy extraction and embedding, tissue sectioning, immunofluorescence, imaging steps and acquisition, and three-dimensional data post-processing of muscle stem cells. • Builds upon the validated method developed by Feng et al. [1], which was optimized for mouse tissue and fills critical gaps in existing literature. • Allows qualitative and quantitative morphological assessment of muscle stem cell nuclei in three-dimensional space. Graphical overview Graphical overview of integrated workflow for three-dimensional visualization of human skeletal muscle stem cells. After the percutaneous muscle biopsy, cut ~25-100 mg of the sample and arrange it according to the desired orientation → Mount sample for sectioning, embed in mounting medium, and freeze in liquid nitrogen-cooled isopentane → Using a cryostat, generate tissue cross-sections in an alternating collection method of 20 μm intervals and place on subbed glass slides → Fix and block sections before incubating in a cocktail of primary antibodies specific for satellite cell nuclei (anti-Pax7) and muscle membrane (anti-laminin) overnight. The following day, incubate sections in the appropriate secondary antibodies (Pax7: goat anti-mouse IgG1 biotin conjugated; laminin: goat anti-rabbit Alexa Fluor 488), apply signal amplification using streptavidin-horseradish peroxidase and tyramide 594 conjugate before counterstaining with DAPI, add mounting media, and coverslip → Using a confocal microscope, search for Pax7 signal, confirm overlap with DAPI adjacent to laminin labeling, apply appropriate laser channels, determine z-stack size, and acquire images in high-pixel-resolution format → For image post-processing, in the software's three-dimensional viewer, modify individual channel histograms to optimize image quality and save as a TIFF.