Ultrastructural Analysis of Enzymatically Dissociated Skeletal Muscle Fibers: Microwave-Assisted Heavy Metal Staining.

In this chapter, we describe a protocol for the preparation of enzymatically dissociated murine skeletal muscle fibers for ultrastructural analysis. Enzymatic dissociation is a widely used method for the isolation of individual muscle fibers. At the same time, this treatment can induce significant physiological alterations, affecting sample characteristics and influencing the outcomes of bioimaging protocols. We found that the conventional transmission electron microscopy (TEM) tissue-processing protocol, performed at room temperature with resin embedding and heavy-metal staining, is unsuitable for samples of this type. Osmium tetroxide and uranyl acetate exhibited a drastically reduced penetration ability in dissociated fibers compared to skeletal muscle fragments or cultured myotubes, leading to a critical reduction in contrast in the resulting electron microscopy images. We used microwave-assisted tissue processing to overcome this obstacle. Besides ultrastructural analysis, the described protocol could be applied to other bioimaging approaches (such as immunostaining and related methods) that require overcoming penetration-related challenges when working with dissociated fibers.