Abstract
The neuromuscular junction (NMJ) is a specialized synapse that connects the terminal end of a motor neuron and a skeletal muscle fiber. Defects in NMJ cause abnormalities of neuromuscular transmission, leading to NMJ disorders. The mammalian diaphragm muscle is essential for respiration and has been widely used to study NMJ formation. Here, we provide a simple and straightforward protocol for preparing diaphragms from embryonic, neonatal, and adult mice and for subsequent NMJ staining.