Background

In the last decades, the nematode Caenorhabditis elegans has emerged as a powerful model organism and has provided an unprecedented opportunity to expand our knowledge in different fields of research, from developmental biology (Mello et al., 1992; Bowerman et al., 1993; Hubbard and Greenstein, 2000), to neuromuscular degeneration (Braungart et al., 2004; McColl et al., 2012; Sorrentino et al., 2017; Romani et al., 2021) and aging (Kimura et al., 1997; Herndon et al., 2002). One of the main reasons that promoted the usage of C. elegans as a laboratory model is that despite its simplicity, it has defined tissues. In particular, it consists of five main tissues: epidermis, reproductive tissues, digestive system, nervous system, and muscle tissue (Spencer et al., 2011). Among these, muscle tissue morphology and function have been widely used to typify C. elegans fitness and healthspan (Fire et al., 1991; Ryu et al., 2016; Fang et al., 2017; D’Amico et al., 2019; Romani et al., 2021). In recent years, numerous protocols have emerged to facilitate the visualization of C. elegans muscles, taking advantage of the transparent nature of this nematode. Phalloidin is a mushroom-derived molecule that specifically binds filamentous actin (F-actin); hence, phalloidin derivatives tagged with fluorescent molecules are commonly used in microscopy to visualize F-actin in different fields of research (Chazotte, 2010). Body wall muscles of the nematode C. elegans possess obliquely striated myofibrils composed of highly organized filaments of actin, whose disposition is altered in pathological conditions (Gieseler et al., 2018). For this reason, phalloidin-based staining of actin filaments has been successfully implemented in nematodes for the detection of cytoskeleton and muscle structure, with important applications in muscle disease and degeneration (Gieseler et al., 2018). However, phalloidin-based protocols typically rely on tedious fixation steps using harmful compounds, such as PFA, and require the preparation of specific, complicated buffers (Costa et al., 1997; Ono, 2001; Bansal et al., 2015; Geisler et al., 2020). We present here a method for C. elegans research that allows phalloidin staining of actin filaments, bypassing fixation, and using conventional buffers. Moreover, by integrating 4’,6-diamidino-2-phenylindole (DAPI) staining in our protocol, we allow the visualization of muscle cell nuclei, whose morphology can be used to interpret cellular homeostasis. Finally, our protocol requires a limited amount of worms and can be suspended at different steps, ensuring a versatile experimental procedure. Overall, our methodology provides a robust, simple, and safer pipeline for the visualization of muscle fibers and nuclei in C. elegans with applications in multiple fields of biomedical research. We predict that this protocol could be coupled with additional dyes specific for other tissues or cellular components, such as endoplasmic reticulum and mitochondria, allowing an even broader overview of worm physiology and homeostasis.