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

Area of Science:

• Biomedical Sciences
• Cell Biology
• Histology

Background:

• Skeletal muscle fiber type composition influences function, metabolism, and disease susceptibility.
• Accurate muscle fiber type analysis is crucial for disease diagnosis and understanding pathophysiology.
• Traditional immunofluorescence (IF) for myosin heavy chain (MyHC) isoforms requires fresh-frozen tissue, posing processing, storage, and biosafety challenges.

Purpose of the Study:

• To present a robust protocol for MyHC labeling on formalin-fixed paraffin-embedded (FFPE) skeletal muscle sections.
• To describe a modified procedure for high-throughput fiber type analysis of intact mouse lower hindlimbs.
• To offer a safer and more stable alternative to traditional fresh-frozen tissue protocols.

Main Methods:

• Developed and validated a protocol for multiplex immunofluorescence (IF) for MyHC labeling on FFPE mouse and human muscle sections.
• Adapted the protocol for analyzing intact mouse lower hindlimb sections, enabling simultaneous multi-muscle analysis.
• Compared the FFPE protocol with traditional fresh-frozen methods regarding sample processing, preservation, and safety.

Main Results:

• The FFPE protocol successfully labels MyHC isoforms in skeletal muscle sections, preserving spatial architecture.
• The modified procedure allows for high-throughput analysis of fiber type composition and morphology across multiple muscles in a single section.
• FFPE embedding streamlines workflows, enhances long-term sample stability, simplifies storage and transport, and improves biosafety by inactivating infectious agents.

Conclusions:

• Formalin-fixed paraffin-embedded (FFPE) skeletal muscle sections provide a robust and practical alternative to fresh-frozen tissues for MyHC-based fiber type analysis.
• This FFPE protocol facilitates easier sample handling, improved preservation, and enhanced safety, making it suitable for clinical and research settings.
• The method is adaptable for various downstream applications, including integration with next-generation techniques favoring FFPE samples.