Optimized multiplex immunofluorescent protocols for simultaneous in situ identification of α-motoneuron subtypes

Accurate identification of α-motoneuron (α-MN) subtypes - slow (S), fast fatigue-resistant (FR), fast fatigue-intermediate (FI), and fast fatigable (FF) - is essential for studying motor circuit organization and selective vulnerability in neurodegenerative disease. While electrophysiological approaches can distinguish these subtypes, existing immunohistochemical (IHC) methods lack the ability to simultaneously identify all four α-MN classes in situ, particularly the FI subtype, limiting their utility for large-scale or tissue-based analyses.

Here, we present novel multiplex immunofluorescent strategies that enables simultaneous in situ identification of S, FR, FI, and FF α-MN subtypes, including intermediate populations, within single sections of mouse lumbar spinal cord. This approach integrates a combinatorial marker framework with optimized co-labeling conditions to resolve subtype-specific molecular signatures, including FI MNs, which have not been previously distinguishable using standard IHC methods.

We establish a systematic validation pipeline demonstrating robust and reproducible subtype classification across multiple protocols, sexes, mouse strains, and disease conditions, including the G93A-SOD mouse model of amyotrophic lateral sclerosis. Labeled populations recapitulate known size distributions and exhibit consistent subtype-specific patterns across lumbar segments, supporting both the accuracy and reproducibility of the method.

By enabling comprehensive in situ classification of all major α-MN subtypes, this approach represents a substantive refinement of multiplex IF, overcoming key limitations of existing IF methods and enabling analyses of α-MN subtype organization and selective vulnerability that were previously not feasible with standard histological techniques. This framework is broadly applicable to studies of motor system organization, aging, and neurodegenerative disease.