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Murine whole-organ immune cell populations revealed by multi-epitope-ligand cartography
Jenny Eckhardt1, Christian Ostalecki, Katarzyna Kuczera
1University Hospital of Erlangen, Erlangen, Germany.
Insights
Multi-epitope-ligand cartography (MELC) enables high-throughput analysis of up to 100 markers on single tissue sections. This study applied MELC to map leukocyte subpopulations in the EAE model, revealing their in situ localization and activation status.
Area of Science:
- Immunology
- Neuroscience
- Microscopy
Background:
- Multi-epitope-ligand cartography (MELC) is an advanced high-throughput fluorescence microscopy technique.
- MELC allows multiplexed staining and imaging of up to 100 markers on a single tissue section.
- Toponomic expression analysis is enabled through pixel-precise image overlay.
Purpose of the Study:
- To adapt MELC for comprehensive analysis of murine leukocyte subpopulations.
- To investigate leukocyte infiltration and activation in the experimental autoimmune encephalomyelitis (EAE) model.
- To correlate MELC findings with quantitative RT-PCR data.
Main Methods:
- Adaptation of MELC for high-throughput analysis of leukocyte subpopulations in murine lymph nodes.
- Application of MELC to non-inflamed and inflamed brain and spinal cord tissues from the EAE model.
- In situ assessment of leukocyte localization and activation status.
Main Results:
- Identification of diverse leukocyte subpopulations, including T cells, dendritic cells, and macrophages.
- Detailed mapping of cellular localization and activation states within EAE tissues.
- Correlation of MELC data with quantitative RT-PCR results.
Conclusions:
- MELC is a powerful tool for dissecting complex immune cell populations in situ.
- The study provides insights into leukocyte dynamics during EAE.
- MELC facilitates comprehensive analysis of immune responses in neurological disease models.
Abstract:
Multi-epitope-ligand cartography (MELC) is an innovative high-throughput fluorescence microscopy-based method. A tissue section is analyzed through a repeated cycling of (1) incubation with a fluorophore-labeled antibody, (2) fluorescence imaging, and (3) soft bleaching. This method allows staining of the same tissue section with up to 100 fluorescent markers and to analyze their toponomic expression using further image processing and pixel-precise overlay of the corresponding images. In this study, we adapted this method to identify a large panel of murine leukocyte subpopulations in a whole frozen section of a peripheral lymph node. Using the resulting antibody library, we examined non-inflamed versus inflamed tissues of brain and spinal cord in the experimental autoimmune encephalomyelitis (EAE) model. The presence and activity of specific leukocyte subpopulations (different T cell subpopulations, dendritic cells, macrophages, etc.) could be assessed and the cellular localizations and the corresponding activation status in situ were investigated. The results were then correlated with quantitative RT-PCR.
