Visualization and Analysis of Neuromuscular Junctions Using Immunofluorescence

The neuromuscular junction (NMJ) is an interface between motor neurons and skeletal muscle fibers, facilitating the transmission of signals that initiate muscle contraction. Its pivotal role lies in ensuring efficient communication between the nervous system and muscles, allowing for precise and coordinated movements essential for everyday activities and overall motor function. To provide insights into neuromuscular disease and development, understanding the physiology of NMJ is essential. We target acetylcholine receptors (AChR) by immunofluorescence assay (IFA) with α-bungarotoxin (BTX; snake venom neurotoxins binding to AChR) to visualize and quantify the NMJ. Changes in AChR distribution or structure can indicate alterations in receptor density, which may be associated with neuromuscular disorders or conditions that affect synaptic transmission. This protocol provides the methodology for isolating and longitudinally sectioning gastrocnemius muscle for AChR-targeted IFA for confocal microscopy and performing quantitative analysis of NMJs. Key features • Visualizes and quantifies NMJs using α-bungarotoxin. • Utilizes high-resolution confocal microscopy for detailed imaging.