Whole-Mount Immunostaining for the Visual Separation of A- and C-Fibers in the Study of the Sciatic Nerve.

Peripheral nerve injuries (PNIs) often result in incomplete functional recovery due to insufficient or misdirected axonal regeneration. Balanced regeneration of myelinated A-fibers and unmyelinated C-fibers is essential for functional recovery, making it crucial to understand their differential regeneration patterns to improve PNI treatment outcomes. However, immunochemical staining does not clearly differentiate between A- and C-fiber axons in whole-mount nerve preparations. To overcome this limitation, we developed a modified protocol by optimizing the immunostaining to restrict the antibody access to myelinated axons. This enables visualization of A-fibers by myelin sheath labeling, while allowing selective staining of unmyelinated C-fiber axons. As a result, A- and C-fibers can be reliably distinguished, facilitating accurate analysis of their regeneration in both normal and post-injury conditions. Combined with confocal microscopy, this approach supports efficient screening of whole-mount nerve preparations to evaluate fiber density, spatial distribution, axonal sprouting, and morphological characteristics. The refined technique provides a robust tool for advancing PNI research and may contribute to the development of more effective therapeutic strategies for nerve repair. Key features • Visual separation of myelinated A-fibers and unmyelinated C-fibers is achieved by restricting the penetration of axon-labeling antibodies through the myelin sheaths. • The protocol also distinguishes A- and C-fibers based on the types of associated Schwann cells. • The protocol is specially designed to distinguish between A- and C-fibers as well as their morphological features in whole-mount nerve preparations. • The protocol does not require specialized reagents, equipment, or techniques, making it highly accessible and reproducible across different research settings.