DEFINING NEURONAL DIVERSITY IN THE DEVELOPING AND ADULT CEREBRAL CORTEX WITH ATLASPLEX TECHNOLOGY

Understanding the cytoarchitecture of the brain is essential for interpreting normal and pathological processes. Although single-cell transcriptomic methodologies have revealed extensive neuronal diversity, these methods lack protein level confirmation and do not capture cellular morphology in situ. Conventional multiplex immunohistochemistry–immunofluorescence (mIHC-IF) approaches further suffer from antibody host-species constraints and weak fluorescence signals.
To overcome these barriers, AtlasPlex mIHC-IF technology was employed to profile neuronal cell types in the developing (E14, E18) and adult mouse cerebral cortex. AtlasPlex combines site-specific biotinylated primary antibodies with tyramide signal amplification (TSA), enabling multiplex detection using same-species antibodies. Comprehensive markers panels integrated transcription factors (PAX6, EMX1, LHX2, TBR1, FOXP2, TBR2, CTIP2, SATB2, CUX1), neurotransmitters (VGluT1, VGAT, GAD2), neuropeptides (VIP, SST), and calcium-binding proteins (CALB2, PVALB, PCP4).
At E14–E18, the 5-plex transcription factor panel (PAX6, EMX1, LHX2, TBR1, TBR2) successfully identified cortical progenitors. In the adult cortex, 3–5 plex panels distinguished glutamatergic (VGluT1) projection neurons and GABAergic interneurons across layers, including layers 2–3 callosal (SATB2, CUX1), layer 5 corticostriatal (SATB2) and corticopontine (BCL11B, PCP4), and layer 6 corticothalamic neurons (FOXP2, BCL11B). GABAergic interneurons (VGAT/GAD2) in layers 2-3 and 5-6 were profiled by VIP, SST, CALB2, and PVALB. Results were consistent with transcriptomic classifications.
AtlasPlex demonstrated robust high-resolution 3–5 plex mIHC-IF performance, enabling precise proteomic profiling of neuronal subtypes in both the developing and adult cerebral cortex. Compatibility with same species antibodies expands panel design and enhances multiplexing capacity, offering a powerful tool for validating transcriptomic data and generating high-resolution neuronal atlases.