Abstract
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.
Highlights
Traditional neuroanatomy immunohistology studies involve low-content analyses of a few antibodies of interest, typically applied and compared across sequential tissue sections
We present a multiplexed fluorescence immunohistochemistry (MP-IHC) labelling and analysis approach for human brain tissue that improves the throughput of conventional IHC and current multiplex protocols through iterative labelling of up to 10 different antibodies per cycle, with no limitation on the number of cycles[8]
Repeated cycles of antibody stripping and relabelling are followed by alignment of images using the DAPI channel from each staining/imaging cycle for spatial registration at the pixel level. We previously demonstrated this method on rodent brain tissue[8], and here we demonstrate the first application of this protocol for human tissue neuroanatomical studies by labelling up to 100 antibodies on formalin-fixed paraffin-embedded sections of human olfactory bulb from neurologically normal, Alzheimer’s (AD), and Parkinson’s disease (PD) patients
Summary
Traditional neuroanatomy immunohistology studies involve low-content analyses of a few antibodies of interest, typically applied and compared across sequential tissue sections. 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. Repeated cycles of antibody stripping and relabelling are followed by alignment of images using the DAPI channel from each staining/imaging cycle for spatial registration at the pixel level We previously demonstrated this method on rodent brain tissue[8], and here we demonstrate the first application of this protocol for human tissue neuroanatomical studies by labelling up to 100 antibodies on formalin-fixed paraffin-embedded sections of human olfactory bulb from neurologically normal ( referred to as ‘no significant pathology’ or NSP), Alzheimer’s (AD), and Parkinson’s disease (PD) patients. MP-IHC provides an efficient and powerful method of conducting such analyses, as the spatial context of all tissue features can be assessed on a single tissue section
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
