Dissection and Whole-Mount Immunofluorescent Staining of Mouse Hind Paw Muscles for Neuromuscular Junction Analysis

Abstract

The neuromuscular junction (NMJ) is a peripheral synaptic connection between a lower motor neuron and skeletal muscle fibre that enables muscle contraction in response to neuronal stimulation. NMJ dysfunction and morphological abnormalities are commonly observed in neurological conditions, including amyotrophic lateral sclerosis, Charcot–Marie–Tooth disease, and spinal muscular atrophy. Employing precise and reproducible techniques to visualise NMJs in mouse models of neuromuscular disorders is crucial for uncovering aspects of neuropathology, revealing disease mechanisms, and evaluating therapeutic approaches. Here, we present a method for dissecting the deep lumbrical and flexor digitorum brevis (FDB) muscles of the mouse hind paw and describe the process of whole-mount immunofluorescent staining for morphological analysis of NMJs. Similar whole-mount techniques have been applied to other muscles, such as the diaphragm; however, dense connective tissue in adult samples often impedes antibody penetration. Moreover, large hind limb muscles, including the gastrocnemius and tibialis anterior, are commonly used to examine NMJs but require embedding and cryosectioning. These additional steps increase the complexity and duration of the protocol and can introduce sectioning artefacts, including transection of NMJs and disruption of morphology. Using small hind paw muscles enables whole-mounting, which completely eliminates the requirement for embedding and cryosectioning. As a result, the entire neuromuscular innervation pattern can be visualised, allowing a more accurate assessment of NMJ development, denervation, and regeneration in mouse models of neurological disease and nerve injury, which can be applied across all postnatal ages.