Traditional neuroanatomy immunohistology studies involve low-content analyses of a few antibodies of interest, typically applied and compared across sequential tissue sections. The efficiency, consistency, and ultimate insights of these studies can be substantially improved using high-plex immunofluorescence labelling on a single tissue section to allow direct comparison of many markers. Here we present an expanded and efficient multiplexed fluorescence-based immunohistochemistry (MP-IHC) approach that improves throughput with sequential labelling of up to 10 antibodies per cycle, with no limitation on the number of cycles, and maintains versatility and accessibility by using readily available commercial reagents and standard epifluorescence microscopy imaging. We demonstrate this approach by cumulatively screening up to 100 markers on formalin-fixed paraffin-embedded sections of human olfactory bulb sourced from neurologically normal (no significant pathology), Alzheimer's (AD), and Parkinson's disease (PD) patients. This brain region is involved early in the symptomology and pathophysiology of AD and PD. We also developed a spatial pixel bin analysis approach for unsupervised analysis of the high-content anatomical information from large tissue sections. Here, we present a comprehensive immunohistological characterisation of human olfactory bulb anatomy and a summary of differentially expressed biomarkers in AD and PD using the MP-IHC labelling and spatial protein analysis pipeline.
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