American Association of Immunologist (AAI) Annual Meeting 2025

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by amyloid-beta plaques, neurofibrillary tangles, neuronal loss, and cognitive impairment. While our understanding of AD has evolved, mapping the cellular and molecular interplay between neurodegeneration and neuroimmunology remains challenging. Recent innovations in spatial biology have enabled multiple advances through the detailed identification of cell lineages and intercellular interactions within the native architecture of many tissue types. In this study, we leveraged a spatial proteomics approach to map the cellular interactions underlying neurodegeneration in AD.

Formalin-fixed paraffin-embedded tissue sections of human AD and healthy brain samples were stained and imaged using the fully-automated sequential immunofluorescence (seqIF™) workflow on COMET™. Images were analyzed by HORIZON™ software to reveal in situ single-cell level features.

We designed a highplex proteomics panel to characterize the spatial localization of AD-associated protein aggregates, along with key cell populations such as neurons, astrocytes, microglia, and vasculature-associated cells in the same tissue section. Our analysis identified inflammatory activation of microglia and astrocytes in the proximity of amyloid-beta plaques. This spatial approach offers novel insights into the spatial relationships between neurodegenerative lesions and the neighboring cells, shedding light on the neuroimmune landscape of AD.

The rise of spatial biology has transformed our ability to investigate tissue structures and cellular interactions at a single-cell resolution. Through techniques such as multiplexed immunofluorescence (mIF) or RNA in situ hybridization (ISH), it is possible to achieve a detailed mapping of key histological and cellular components along with their activation states. Combining those two technologies on the same tissue section could allow for better profiling of complex tissues like the mouse brain.

Here, using the COMET™ staining and imaging platform, RNAscope™ HiPlex Pro and sequential immunofluorescence (seqIF™) assays were integrated and fully automated for same-section RNA and protein detection on fixed-frozen mouse brain tissues. Through precise temperature control and gentle reagent distribution, fragile frozen sections can now be used to visualize up to a 12-plex RNA and 24-plex protein panel. Using this newly developed multiomics assay, we designed a panel to deepen the understanding of normal brain function and architecture through mapping of molecular networks and by studying the interactions between major brain cell types (e.g., neurons, astrocytes, oligodendrocytes) and different immune cells (e.g., microglia and macrophages).

Our results highlight the compatibility of combined RNAscope™ HiPlex Pro and seqIF™ workflows on the COMET™ with frozen sections, while showing valuable insights into the interactions between the immune and nervous system.

R&D Systems protein experts have made a breakthrough in cell culture optimization with proteins designed for performance. These proteins have tailored functionalities such as hyperactivity, improved receptor affinities, and increased heat stability. Paving the way for spatial multiomics, the industry-leading RNAscope™ platform now offers novel protease-free workflows that enable high-accuracy, single-cell insights into cell phenotype and function, from discovery to therapeutic development.

Multiple sclerosis (MS) is a neurological disorder characterized by autoimmune-driven demyelination and neurodegeneration of the central nervous system (CNS). The complexity of the immune response and intricated interactions within the CNS microenvironment remain challenging to pinpoint. Recent advancements in spatial biology offer powerful tools to investigate MS lesions. In this study, we leverage automated multiplex immunofluorescence to investigate protein expression and cellular relationships in the brain of MS patients.

Formalin-Fixed Paraffin-Embedded tissue sections of human MS and normal brain samples were stained and imaged on COMET™, a fully automated platform for sequential immunofluorescence (seqIF™) assays.

We designed a high-plex immunofluorescence panel to assess spatial alterations occurring in MS lesions. We focused on the characterization of neuroinflammation by leveraging the simultaneous detection of multiple microglia and astrocyte biomarkers on a single slide, together with neurons and vasculature components. Our data confirmed the occurrence of the local immune response associated with MS, providing insights into the disease-specific alterations at a spatially resolved level.

The automated high-plex assay used in this study significantly reduces the consumption of precious tissue samples, while enabling a comprehensive analysis of the brain architecture in neurological disorders to gain a deeper understanding of complex mechanisms of neurodegeneration.