Adaptable Immunofluorescence Protocol for Muscle Fiber Typing in FFPE Human and Mouse Skeletal Muscle and Intact Mouse Hindlimbs.

Skeletal muscle fiber type composition affects muscle function, metabolism, and disease vulnerability. In addition, muscle fiber type analysis informs disease diagnosis and underlying pathophysiology. Multiple methodologies can be used to assess muscle fiber type; however, immunofluorescence (IF) for myosin heavy chain (MyHC) isoforms is the most widely used modern approach due to its relative ease, time-effectiveness, single-cell resolution, and capacity to preserve spatial positioning within the native tissue architecture. Here, we present a protocol for IF for MyHC labeling on formalin-fixed paraffin-embedded (FFPE) mouse and human muscle sections. We then describe a modified procedure for fiber type analysis of the intact mouse lower hindlimb, enabling high-throughput muscle composition and morphological analysis across distinct muscles on a single tissue section. Traditionally, IF labeling for MyHC isoforms required fresh tissue flash-frozen in liquid nitrogen-cooled isopentane, which, while effective, presents challenges for sample processing and preservation, long-term storage, transport, and biosafety. Comparatively, embedding tissue in paraffin after formalin fixation streamlines clinical workflows, preserves morphology, improves long-term sample stability, and simplifies sample storage and transport. Furthermore, FFPE effectively inactivates most infectious agents, which can be retained in frozen sections. Thus, FFPE samples are typically safe for standard laboratory handling and are not classified as biohazardous. This approach can be adapted for use with a range of downstream applications, including integration of fiber type analysis with emerging next-generation techniques that favor FFPE samples. In sum, this method offers a robust alternative to traditional fresh-frozen protocols and allows for simultaneous fiber type analysis across multiple muscle tissues. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Multiplex Immunofluorescence for MyHC Labeling in FFPE Skeletal Muscle Tissues Alternate Protocol 1: Immunofluorescence for MyHC 2x Labeling in FFPE Skeletal Muscle Tissues Alternate Protocol 2: Multiplex Immunofluorescence for MyHC Labeling in FFPE Whole Hindlimb Sections.