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.