High-Resolution Skeletal Muscle Fiber Typing Using Four-Color Immunofluorescence Microscopy.

Skeletal muscle is composed of heterogeneous fiber types defined by distinct myosin heavy chain (MYH) isoforms. Accurate fiber type identification is essential for studying skeletal muscle development, plasticity, disease, and intervention responses. Traditional fiber type classification techniques are limited by either lack of specificity or the inability to simultaneously detect all fiber types in a single section. Here, we describe a novel four-channel immunofluorescent staining protocol that enables simultaneous visualization and quantification of type I, IIA, IIX, and IIB fibers within a single cross-section of rodent skeletal muscle. This high-resolution method improves throughput and eliminates the need for serial sectioning but also allows for the identification of hybrid fibers. Moreover, membrane staining can be applied to simultaneously perform cross-sectional area analysis. This approach enhances the precision and efficiency of skeletal muscle phenotyping in both physiological and pathological contexts by capturing the full spectrum of skeletal muscle fibers in a single image, thus providing a powerful tool for investigating fiber type-specific development and adaptation.