MAPPING ASTROCYTE HETEROGENEITY IN THE MOUSE BRAIN USING AN RNA–PROTEIN MULTIOMICS ASSAY

Astrocytes are critical regulators of central nervous system (CNS) function, contributing to synaptic homeostasis, metabolic support, and neuroinflammation. Their phenotypic and functional heterogeneity underlies region-specific roles in brain development and disease. To explore astrocytic heterogeneity and neuron-glia interactions in the mouse brain, we implemented a spatial multiomics workflow integrating RNA in situ hybridization and immunofluorescence.

Here, we introduce a protease-free workflow for the RNAscope™ multiplex fluorescent v2 assay, combining RNA in situ hybridization (ISH) with protein immunofluorescence to detect both RNA and protein markers simultaneously on the same frozen mouse brain tissue sections. This manual multiplex protease-free workflow enables the visualization of RNA and protein targets without enzymatic digestion, preserving tissue morphology and antigenicity.

Using this workflow, we profiled canonical astrocyte markers—Slc1a3 (encoding GLAST), Aldh1l1, and Glul (encoding glutamine synthetase) and co-localized them with glial fibrillary acidic protein (GFAP), a pan astrocytic marker. To examine spatial associations between astrocytes and neurons, we co-detected Gja1 mRNA (encoding the gap junction protein connexin-43 in astrocytes) alongside MAP2 protein, a neuronal dendritic marker.

This spatial multiomics approach enables detection of variation in astrocyte gene expression and subtype distribution, highlighting diverse functional states within the astrocyte lineage. Importantly, our protease-free method preserved delicate cellular features, enabling accurate co-detection of molecular signals that inform astrocyte identity and their interactions with neurons.

RNAscope™ manual multiplex assay, provides a robust tool for dissecting cell-type–specific molecular landscapes, facilitating deeper insights into glial biology across development, aging, and neurodegeneration.