Combined single-molecule fluorescence in situ hybridization and immunohistochemistry analysis in intact murine dorsal root ganglia and sciatic nerve

Xinbei Li; Sreenivas Eadara; Sangmin Jeon; Yan Liu; Gabriella Muwanga; Lintao Qu; Michael J. Caterina; Mollie K. Meffert

STAR Protocols (Jun 2021)

Combined single-molecule fluorescence in situ hybridization and immunohistochemistry analysis in intact murine dorsal root ganglia and sciatic nerve

Xinbei Li,
Sreenivas Eadara,
Sangmin Jeon,
Yan Liu,
Gabriella Muwanga,
Lintao Qu,
Michael J. Caterina,
Mollie K. Meffert

Affiliations

Xinbei Li: Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
Sreenivas Eadara: Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
Sangmin Jeon: Department of Neurosurgery and Neurosurgery Pain Research Institute, Baltimore, MD 21205, USA
Yan Liu: Department of Neurosurgery and Neurosurgery Pain Research Institute, Baltimore, MD 21205, USA
Gabriella Muwanga: Department of Neurosurgery and Neurosurgery Pain Research Institute, Baltimore, MD 21205, USA
Lintao Qu: Department of Neurosurgery and Neurosurgery Pain Research Institute, Baltimore, MD 21205, USA
Michael J. Caterina: Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Neurosurgery and Neurosurgery Pain Research Institute, Baltimore, MD 21205, USA; Solomon H. Snyder Department of Neuroscience, Baltimore, MD 21205, USA
Mollie K. Meffert: Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Solomon H. Snyder Department of Neuroscience, Baltimore, MD 21205, USA; Corresponding author

Journal volume & issue: Vol. 2, no. 2
p. 100555

Abstract

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Summary: Single-molecule fluorescence in situ hybridization (smFISH) allows spatial mapping of gene expression. This protocol presents advances in smFISH fidelity and flexibility in intact murine sensory nervous system tissue. An approach using RNAscope probes allows multiplexing, enhanced target specificity, and immunohistochemistry compatibility. Computational strategies increase quantification accuracy of mRNA puncta with a point spread function for clustered transcripts in the dorsal root ganglion and 3D masking for intermingled sciatic nerve cell types. Approaches are validated for mRNAs of modest (Lin28a) and medium (Ppib) steady-state abundance in neurons.

Published in STAR Protocols

ISSN: 2666-1667 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Science (General)
Website: https://star-protocols.cell.com/

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