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

doi:10.1016/j.xpro.2021.100555

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

School of Medicine

Research output: Contribution to journal › Article › peer-review

Abstract

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.

Original language	English (US)
Article number	100555
Journal	STAR Protocols
Volume	2
Issue number	2
DOIs	https://doi.org/10.1016/j.xpro.2021.100555
State	Published - Jun 18 2021

Keywords

In Situ Hybridization
Microscopy
Molecular Biology
Molecular/Chemical Probes
Neuroscience

ASJC Scopus subject areas

Neuroscience(all)
Biochemistry, Genetics and Molecular Biology(all)
Immunology and Microbiology(all)

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10.1016/j.xpro.2021.100555

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

abstract = "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.",

keywords = "In Situ Hybridization, Microscopy, Molecular Biology, Molecular/Chemical Probes, Neuroscience",

author = "Xinbei Li and Sreenivas Eadara and Sangmin Jeon and Yan Liu and Gabriella Muwanga and Lintao Qu and Caterina, {Michael J.} and Meffert, {Mollie K.}",

note = "Funding Information: We thank members of the Meffert and Caterina laboratories for helpful discussions and critical feedback on the manuscript and Julie Gohkale for assistance with pilot RNAScope experiments. This work was funded by NINDS R01NS103974 and a Discovery Fund Synergy Award to M.J.C. and M.K.M.; a Braude Foundation award to M.K.M.; NIH R25GM109941 to G.M.; and NINDS NS050274 to the Hopkins Neuroscience MPI core. Publisher Copyright: {\textcopyright} 2021 The Author(s)",

year = "2021",

month = jun,

day = "18",

doi = "10.1016/j.xpro.2021.100555",

language = "English (US)",

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T1 - Combined single-molecule fluorescence in situ hybridization and immunohistochemistry analysis in intact murine dorsal root ganglia and sciatic nerve

AU - Li, Xinbei

AU - Eadara, Sreenivas

AU - Jeon, Sangmin

AU - Liu, Yan

AU - Muwanga, Gabriella

AU - Qu, Lintao

AU - Caterina, Michael J.

AU - Meffert, Mollie K.

N1 - Funding Information: We thank members of the Meffert and Caterina laboratories for helpful discussions and critical feedback on the manuscript and Julie Gohkale for assistance with pilot RNAScope experiments. This work was funded by NINDS R01NS103974 and a Discovery Fund Synergy Award to M.J.C. and M.K.M.; a Braude Foundation award to M.K.M.; NIH R25GM109941 to G.M.; and NINDS NS050274 to the Hopkins Neuroscience MPI core. Publisher Copyright: © 2021 The Author(s)

PY - 2021/6/18

Y1 - 2021/6/18

N2 - 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.

AB - 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.

KW - In Situ Hybridization

KW - Microscopy

KW - Molecular Biology

KW - Molecular/Chemical Probes

KW - Neuroscience

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JO - STAR Protocols

JF - STAR Protocols

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ER -

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

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Keywords

ASJC Scopus subject areas

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