Romani Mario, Auwerx Johan
Laboratory of Integrative Systems Physiology, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
Bio Protoc. 2021 Oct 5;11(19):e4183. doi: 10.21769/BioProtoc.4183.
Advances in research have allowed scientists to recapitulate different human disorders, from neurodegenerative diseases to muscle dysfunction, in these nematodes. Concomitantly, the interest in visualizing organs affected by these conditions has grown, leading to the establishment of different antibody- and dye-based staining protocols to verify tissue morphology. In particular, the quality of muscle tissue has been largely used in nematodes as a readout for fitness and healthspan. Phalloidin derivatives, which are commonly used to stain actin filaments in cells and tissues, have been implemented in the context of research for visualization of muscle fibers. However, the majority of the phalloidin-based protocols depend on fixation steps using harmful compounds, preparation of specific buffers, and large amounts of worms. Herein, we implemented a safer and more flexible experimental procedure to stain actin filaments in using phalloidin-based dyes. Lyophilization of the worms followed by their acetone permeabilization allows bypassing the fixation process while also providing the opportunity to suspend the experiment at different steps. Moreover, by using conventional buffers throughout our protocol, we avoid the additional preparation of solutions. Finally, our protocol requires a limited number of worms, making it suitable for slow-growing strains. Overall, this protocol provides an efficient, fast, and safer method to stain actin filaments and visualize muscle fibers in . Graphic abstract: Schematic overview of phalloidin staining in for assessing muscle fiber morphology.
研究的进展使科学家能够在这些线虫中重现从神经退行性疾病到肌肉功能障碍等不同的人类疾病。与此同时，人们对可视化受这些疾病影响的器官的兴趣日益浓厚，这导致建立了不同的基于抗体和染料的染色方案来验证组织形态。特别是，肌肉组织的质量在很大程度上已被用作线虫健康和寿命的指标。鬼笔环肽衍生物通常用于对细胞和组织中的肌动蛋白丝进行染色，已被应用于线虫研究中以可视化肌肉纤维。然而，大多数基于鬼笔环肽的方案依赖于使用有害化合物的固定步骤、特定缓冲液的制备以及大量的线虫。在此，我们实施了一种更安全、更灵活的实验程序，使用基于鬼笔环肽的染料对线虫中的肌动蛋白丝进行染色。线虫冻干后进行丙酮通透处理，这样可以绕过固定过程，同时也提供了在不同步骤暂停实验的机会。此外，在整个实验方案中使用常规缓冲液，我们避免了额外的溶液制备。最后，我们的方案所需的线虫数量有限，使其适用于生长缓慢的线虫菌株。总体而言，该方案提供了一种有效、快速且更安全的方法来对线虫中的肌动蛋白丝进行染色并可视化肌肉纤维。图形摘要：用于评估肌肉纤维形态的线虫鬼笔环肽染色示意图。
