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

Research output: Contribution to journalArticlepeer-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 languageEnglish (US)
Article number100555
JournalSTAR Protocols
Volume2
Issue number2
DOIs
StatePublished - Jun 18 2021
Externally publishedYes

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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