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

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

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 sp...

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Bibliographic Details
Main Authors: Xinbei Li, Sreenivas Eadara, Sangmin Jeon, Yan Liu, Gabriella Muwanga, Lintao Qu, Michael J. Caterina, Mollie K. Meffert
Format: Article
Language:English
Published: Elsevier 2021-06-01
Series:STAR Protocols
Subjects:
Microscopy
Molecular Biology
In Situ Hybridization
Neuroscience
Molecular/Chemical Probes
Online Access:http://www.sciencedirect.com/science/article/pii/S2666166721002628
Description
Summary: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.
ISSN:2666-1667

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