Satellite cells are the resident stem cells of skeletal muscle, located on the surface of a myofibre, beneath the surrounding basal lamina. Satellite cells are responsible for the homeostasis, hypertrophy and repair of skeletal muscle fibres, being activated to enter proliferation and generate myoblasts that either fuse to existing myofibres, or fuse together for de novo myofibre formation. Isolating muscle fibres allows the associated satellite cells to be obtained while remaining in their anatomical niche beneath the basal lamina, free of interstitial and vascular tissue. Myofibres can then be immunostained to examine gene expression in quiescent satellite cells, or cultured to activate satellite cells before immunostaining to investigate gene expression dynamics during myogenic progression and self-renewal. Here, we describe methods for the isolation, culture and immunostaining of muscle fibres for examining satellite cell biology.