Optimized manual multiplex immunofluorescence protocol for human nasal tissue

The human nose is a complex sensory organ responsible for air filtration, moisture regulation, heat exchange, and odor detection. To resolve its cellular and structural heterogeneity-including regions traditionally associated with respiratory and olfactory functions-we applied multiround multiplex immunofluorescence to human postmortem nasal samples. We optimized a manual workflow combining autofluorescence quenching and efficient antibody stripping for frontal nasal and olfactory bulb sections. Multiple published protocols were systematically tested and adapted, with particular emphasis on preserving tissue integrity at bone-soft tissue interfaces. Using this approach, we performed multiplex staining on full nasal sections with five antibodies across three rounds, followed by a validation round with GFAP. Marker panels included K5 (basal cells), TUBB3 (neuronal elements), ANXA1 and E-cadherin (epithelial borders), COLIV (basement membrane), and GFAP (glial/ensheathing cells). This optimized method enables spatial mapping of distinct cell types within intact human nasal tissue and provides a robust platform for future studies on epithelial organization and structural remodeling in health and disease.