A Novel Optical Tissue Clearing Protocol for Mouse Skeletal Muscle to Visualize Endplates in Their Tissue Context
A Novel Optical Tissue Clearing Protocol for Mouse Skeletal Muscle to Visualize Endplates in Their Tissue Context
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DOI：
10.3389/fncel.2019.00049
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发表时间：
2019-02-27
影响因子：
5.3
通讯作者：
Rudolf, Ruediger
中科院分区：
文献类型：
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作者：
Williams, Marion Patrick Ivey;Rigon, Matteo;Rudolf, Ruediger
神经肌肉连接介导骨骼肌收缩，当其形态和功能受损时，在几种神经肌肉疾病中发挥重要作用。然而，由于它们的体积小，分布稀疏，分布在相对较大的、固有不透明的肌肉组织中，因此对NMJ形态的分析仅限于戏弄纤维制剂、纵向肌肉切片和平面肌肉。因此，在实验范例中经常使用的肌肉类型中，对NMJ形态、数量、分布和分配的全组分析也不可能确定。由于缺乏与清晰和持久的NMJ污渍兼容的光学组织清除技术，这种不可能性进一步加剧。在这里，我们提出了一种新颖的、高度可重复的肌肉组织清理方案。基于基于水凝胶的组织清除方法，该方案允许标记和检测野生型和患病小鼠成年后肢指长伸肌中的所有NMJs。该方法也适用于成年小鼠膈肌，可用于不同的染色剂，包括毒素、凝集素、抗体和核染料。这将有助于了解后肢肌肉全坐骑NMJs的分布、形态特征和肌肉组织背景，为生物医学和基础研究提供依据。
Neuromuscular junctions (NMJs) mediate skeletal muscle contractions and play an important role in several neuromuscular disorders when their morphology and function are compromised. However, due to their small size and sparse distribution throughout the comparatively large, inherently opaque muscle tissue the analysis of NMJ morphology has been limited to teased fiber preparations, longitudinal muscle sections, and flat muscles. Consequently, whole mount analyses of NMJ morphology, numbers, their distribution, and assignment to a given muscle fiber have also been impossible to determine in muscle types that are frequently used in experimental paradigms. This impossibility is exacerbated by the lack of optical tissue clearing techniques that are compatible with clear and persistent NMJ stains. Here, we present MYOCLEAR, a novel and highly reproducible muscle tissue clearing protocol. Based on hydrogel-based tissue clearing methods, this protocol permits the labeling and detection of all NMJs in adult hindleg extensor digitorum longus muscles from wildtype and diseased mice. The method is also applicable to adult mouse diaphragm muscles and can be used for different staining agents, including toxins, lectins, antibodies, and nuclear dyes. It will be useful in understanding the distribution, morphological features, and muscle tissue context of NMJs in hindleg muscle whole mounts for biomedical and basic research.