Protocol for applying expansion microscopy to the study of mammalian neuromuscular junctions

Expansion microscopy (ExM) is a tissue-swelling technique that enables super-resolution imaging through a specialized preparation process that physically expands stained biomolecules within a fixed sample. Here, we detail a protocol to apply the 4× ExM technique to neuromuscular junctions (NMJs) from both human and mouse muscle preparations. We describe steps for muscle fixation, microdissection, staining, gelation, and digestion. We then detail procedures for expansion, mounting, imaging, analysis, and quantification. This protocol can be used to reveal nanoscale anatomical NMJ features. For complete details on the use and execution of this protocol, please refer to Ramadan et al.¹.