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Method Article
Mark Bathe
Bathe Lab, Department of Biological Engineering, MIT, Cambridge, MA, USA
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Neuronal synapses contain hundreds of protein species whose expression levels and sub-micron-scale localizations are core determinants of neuronal plasticity and signal transmission. The spectral overlap of fluorophores used in conventional fluorescence microscopy limits the number of labeled species to four cellular targets within a given sample. To overcome the spectral limit, we introduce LNA-PRISM1: Locked Nucleic Acid Probe-based Imaging for Sequential Multiplexing. In contrast to DNA-PAINT2,3 (Points Accumulation In Nanoscale Topography), LNA-PRISM utilizes high affinity LNA probes and DNA-barcoded cellular target markers to realize fast, multiplexed diffraction-limited confocal imaging in the same sample. In this protocol, we describe the general strategies for antibody and probe conjugation, immunostaining, and sequential imaging for 13 neuronal targets focusing on synaptic proteins. LNA-PRISM offers faster multiplexed confocal imaging and lower risk of sample and epitope disruption with mild buffer exchange compared to approaches based on sequential antibody staining-elution. Our approach is broadly applicable to other cell culture and scalable to dozens of target proteins. It is compatible with high-content screening platforms commonly used to interrogate the phenotypic impact of genetic and drug perturbations.
Keywords
Multiplexed imaging, Neuronal synapses, Nucleic acid probe-based imaging, Confocal microscopy, Locked nucleic acid, synapses, Probe-based Imaging for Sequential Multiplexing
The authors declare no competing financial interests
This is a list of supplementary files associated with this preprint. Click to download.
- supplement0.docx
Table 1 Antibody information.
- supplement0.docx
Table 2 Reagent information.
- supplement0.docx
Table 3 Docking strand and imaging probe sequences.
- supplement0.docx
Table 4 Buffer compositions.
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Associated Publications
Method Article
Mark Bathe
Bathe Lab, Department of Biological Engineering, MIT, Cambridge, MA, USA
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 14 Jun, 2018
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Neuronal synapses contain hundreds of protein species whose expression levels and sub-micron-scale localizations are core determinants of neuronal plasticity and signal transmission. The spectral overlap of fluorophores used in conventional fluorescence microscopy limits the number of labeled species to four cellular targets within a given sample. To overcome the spectral limit, we introduce LNA-PRISM1: Locked Nucleic Acid Probe-based Imaging for Sequential Multiplexing. In contrast to DNA-PAINT2,3 (Points Accumulation In Nanoscale Topography), LNA-PRISM utilizes high affinity LNA probes and DNA-barcoded cellular target markers to realize fast, multiplexed diffraction-limited confocal imaging in the same sample. In this protocol, we describe the general strategies for antibody and probe conjugation, immunostaining, and sequential imaging for 13 neuronal targets focusing on synaptic proteins. LNA-PRISM offers faster multiplexed confocal imaging and lower risk of sample and epitope disruption with mild buffer exchange compared to approaches based on sequential antibody staining-elution. Our approach is broadly applicable to other cell culture and scalable to dozens of target proteins. It is compatible with high-content screening platforms commonly used to interrogate the phenotypic impact of genetic and drug perturbations.
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