A triple-immunohistochemistry protocol for axonal characterization and quantification in human nervous tissue

Precise characterization of axonal subtypes within human nervous tissue is essential for studies of nerve composition, injury, regeneration, and clinical applications. However, the composition and spatial distribution of axonal populations within human nerves remain incompletely characterized. Most immunohistochemical approaches primarily rely on double-labeling strategies, limiting the simultaneous assessment of multiple axonal populations and often requiring separate tissue sections for complete characterization. To address these limitations, a reproducible triple-immunofluorescence protocol was established for human nervous tissue, enabling the identification and quantification of multiple axonal subtypes within the same tissue section. Two triple-staining combinations were employed targeting (1) neurofilament (NF), choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) for differentiation of sensory, motor and sympathetic axons, and (2) NF, TH and myelin basic protein (MBP) for the assessment of unmyelinated sympathetic and myelinated non-sympathetic fibers. Confocal microscopy combined with a standardized image-analysis workflow enabled reproducible quantification of axonal populations. In addition, a robust method for quantifying thin TH-positive axons was developed to minimize time-consuming manual quantification. This protocol enables reliable differentiation and quantitative analysis of sensory, motor, sympathetic, and myelinated axonal populations and provides a comprehensive overview of nervous tissue composition, while remaining time- and resource-efficient. This method may facilitate future research of nerve composition, organization and pathology.