Optimizing Glyoxal Fixation for Preserving Quiescence in Muscle Stem Cells

Abstract

Preservation and healing of muscle structures have been shown to be attributed to a pool of undifferentiated myogenic precursors known as satellite cells or Muscle stem cells (MuSCs). These cells exist in a resting state called quiescence and are activated in response to injury to either self-renew or initiate the myogenic differentiation program. This ability to balance regeneration and repair is essential for muscle homeostasis. Although quiescence is a dynamic and tightly controlled state, the molecular mechanisms that sustain it remain poorly understood. A key challenge in studying quiescent MuSCs is their rapid activation during tissue isolation, which results in the loss of their native molecular features. To preserve their in vivo state, we employed in situ chemical fixation prior to isolation. Selecting the appropriate fixative was critical, as it affects both the preservation of molecular structures and the compatibility with downstream analyses. Paraformaldehyde (PFA) is widely used due to its capacity to halt cellular processes, but it forms extensive crosslinks between nucleic acids and proteins, often compromising RNA extraction and antibody access. To address these limitations, we tested glyoxal, a dialdehyde fixative known for inducing milder crosslinking. We found that glyoxal effectively arrests transcriptional activation in MuSCs while preserving RNA quality and epitope accessibility compared to PFA. We evaluated glyoxal fixation for compatibility with RNA extraction and sequencing in MuSCs and established a preliminary workflow for isolating and analyzing MuSCs from fixed muscle tissue. While glyoxal fixation preserved RNA quality and prevented activation in C2C12 cells, RNA extracted from fixed MuSCs showed lower yield and quality compared to live-sorted controls, suggesting that further optimization is required to fully enable transcriptomic analysis from fixed tissues. This approach supports various downstream applications, including western blot, qRT-PCR, and immunofluorescence. By preserving the native state of quiescent MuSCs, our method offers a reliable framework for studying their molecular features, such as alternative splicing and post-transcriptional regulation. While much attention has been given to activated or proliferating stem cells, quiescence remains relatively understudied. This work helps bridge that gap and opens new avenues for investigating the biology of muscle stem cell quiescence.