A Detailed Workflow For Three-dimensional Visualization Of Human Skeletal Muscle Stem Cell Nuclei

Muscle stem cells, also known as satellite cells (SCs), can repair damaged muscle fibers following intense physical activity. Days after damage, SCs activate, proliferate, and differentiate into muscle cells, regenerating the damaged tissue. Activated SCs have been shown to increase in size which may influence their function. Previous research has captured images of activated SCs expanding in two-dimensions (2D; x,y); however, there has yet to be a method for identifying SCs in three-dimensional space (3D; x,y,z) to look at nuclear morphology. PURPOSE: To generate a detailed workflow for visualizing human skeletal muscle SC nuclei in 3D space to visualize nuclear morphology. METHODS: A human muscle biopsy from the vastus lateralis was obtained, embedded in liquid nitrogen-cooled isopentane, and stored at -80 °C. Cross-sections were then generated at 20 μm and collected on glass slides before immunohistochemical labeling of SCs. Briefly, sections were fixed, blocked, and incubated overnight in primary antibody against Pax7 to label quiescent SCs. Sections were then rinsed, and incubated in AlexaFluor 594 secondary antibody, before signal amplification with tyramide reagent and nuclear counterstaining with DAPI. Slides were coverslipped and imaged with a Leica TCS SPE Laser Scanning Confocal DM6-Q upright microscope. RESULTS: 3D images in a rotating sequence of two SCs labeled with a Pax7 primary antibody can be visualized in Figure 1. CONCLUSION: After muscle-damaging exercise, activated SCs can possibly change size and shape. Following the methods above, SC nuclei can be visualized in 3D space to observe these morphological characteristics.Supported by Dr. Eduardo Rosa-Molinar, work completed in the Microscopy & Analytical Imaging Research Resource Core Lab