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Related Experiment Video

Updated: Jun 12, 2025

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture
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Profiling lamina specific pyramidal neurons using postmortem human formalin fixed paraffin embedded frontal cortex

Aleksandra Stanisavljevic1, Kyrillos W Ibrahim1, Philip H Stavrides1

  • 1Center for Dementia Research, Nathan Kline Institute, Orangeburg, NY, USA.

Journal of Neuroscience Methods
|June 5, 2025
PubMed
Summary

Digital spatial profiling (DSP) allows RNA sequencing of specific neurons in human brain tissue. This method requires less tissue and provides high-resolution data for detailed analysis of brain cells.

Keywords:
Digital spatial profilingFFPEFrontal cortexGeoMxPyramidal neurons

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Area of Science:

  • Neuroscience
  • Genomics
  • Molecular Biology

Background:

  • Digital spatial profiling (DSP) is an advanced technique for RNA sequencing (RNA-seq) in neuronal populations.
  • DSP links RNA-seq data to spatially defined samples using tissue-bound probes.
  • The GeoMx DSP system was utilized for spatial transcriptomic analysis of human postmortem brain tissue.

Purpose of the Study:

  • To establish a protocol for DSP using human postmortem formalin-fixed paraffin-embedded (FFPE) frontal cortex tissue.
  • To enable spatial characterization of lamina-specific pyramidal neurons and cortical ribbons.
  • To identify Layer III (L3) and Layer V (L5) pyramidal neurons using a specific neuronal marker.

Main Methods:

  • Protocol development for DSP on human postmortem FFPE frontal cortex.
  • Identification and selection of L3 and L5 pyramidal neurons from Brodmann area 9.
  • Utilizing the GeoMx DSP system for probe collection and transcriptomic analysis.

Main Results:

  • Significantly reduced FFPE tissue requirement for robust single-population RNA-seq.
  • Demonstrated that ~20 pyramidal neurons or one lamina-specific cortical ribbon from a single 5 µm section yields robust RNA-seq reads.
  • Bioinformatic analysis revealed similarities and differences between single neurons and admixed cell types in cortical ribbons.

Conclusions:

  • The optimized DSP assay delivers high-resolution RNA-seq data.
  • Demonstrated the utility and versatility of the GeoMx platform for analyzing individual neurons and cortical ribbons.
  • The method is suitable for downstream analyses of postmortem FFPE human brain tissue.