Background

Investigation of neuromuscular junction (NMJ) morphology by immunochemistry can provide important insights into the physiological and pathological status of neuromuscular disorders. Sectioning and muscle fiber tearing are commonly required to prepare experimentally accessible samples, while muscles that are flat and thin can be investigated with whole-mount immunohistochemistry for a comprehensive overview of the entire innervation pattern. The diaphragm is important for respiratory function and one of the flat muscles frequently used for studying neuromuscular development as well as neuromuscular pathology. Nevertheless, techniques for reliable whole-mount immunolabeling of adult diaphragms are lacking, mainly due to the poor tissue permeability of labeling reagents. An effective approach for researchers to be able to comprehensively visualize and characterize NMJ defects of the adult diaphragm in mouse models is therefore of clear importance.

New Method

This protocol demonstrates that the diaphragm can be thinned and spread out under even pressure using two Perspex boards for better whole-mount immunostaining.

Results

The expanded mouse diaphragm allows the comprehensive assessment of a number of NMJ phenotypes.

Comparison with existing methods

Most peer-reviewed and online protocols can be applied to the embryonic diaphragm but fail to show the entire innervation pattern in the adult diaphragm. Our method provides a convenient approach and present a clear innervation pattern that increases the reliability of the assessment of NMJ phenotypes in the diaphragm.

Conclusions

This simple method for whole-mount immunostaining of the adult diaphragm will allow researchers to perform a detailed analysis of the neuromuscular system in mouse models.

中文翻译：

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用夹心样装置对成年小鼠diaphragm肌神经肌肉接头进行整装染色

背景

通过免疫化学研究神经肌肉接头（NMJ）的形态可以为神经肌肉疾病的生理和病理状态提供重要的见解。通常需要进行切片和肌纤维撕裂以制备实验上可接近的样品，而平坦和稀薄的肌肉可以用整装免疫组织化学法进行研究，以全面了解整个神经支配模式。隔膜对于呼吸功能很重要，是经常用于研究神经肌肉发育以及神经肌肉病理学的扁平肌之一。然而，主要是由于标记试剂的组织渗透性差，因此缺乏可靠的对成人whole肌进行全安装免疫标记的技术。

新方法

该协议表明，可以使用两个Perspex板在均匀压力下将隔膜变薄并展开，以实现更好的整装免疫染色。

结果

扩大的小鼠横diaphragm膜可以全面评估许多NMJ表型。

与现有方法的比较

大多数经过同行评审和在线审查的协议都可以应用于胚胎diaphragm肌，但无法显示成年diaphragm肌的整个神经支配模式。我们的方法提供了一种方便的方法，并提供了清晰的神经支配模式，从而提高了评估diaphragm肌NMJ表型的可靠性。

结论

这种简单的成年diaphragm肌全程免疫染色方法将使研究人员能够对小鼠模型中的神经肌肉系统进行详细分析。