Simultaneous Detection of RNAs and Proteins with Subcellular Resolution

Methods Mol Biol. 2020:2161:59-73. doi: 10.1007/978-1-0716-0680-3_6.

Abstract

We describe the detailed methods of "immunoFISH" to analyze the expression level and the spatial localization of RNA transcripts and proteins on cultured cells and formal-fixed, paraffin-embedded (FFPE) tissue sections. On cultured cells, we detect specific transcripts using the Stellaris fluorescence in situ hybridization (FISH) probes labeled with fluorophores that target multiple regions along the desired transcripts and proteins combining the immunofluorescent staining. On FFPE tissue sections, we use the RNAscope FISH probes, modified branched DNA (bDNA) probes to amplify the RNA signals, followed by immunofluorescent staining for protein detection. The abundance, composition, and spatial distribution are determined by signals from fluorescently labeled proteins and individual transcripts of images acquired using high-resolution fluorescence microscopy.

Keywords: Immunofluorescent; Subcellular localization; immunoFISH; smFISH.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line, Tumor
  • Cells, Cultured
  • Fluorescent Dyes / chemistry
  • Humans
  • In Situ Hybridization, Fluorescence / methods*
  • In Situ Hybridization, Fluorescence / standards
  • Limit of Detection
  • Proteins / metabolism
  • RNA, Messenger / metabolism
  • Staining and Labeling / methods
  • Tissue Embedding / methods
  • Tissue Fixation / methods

Substances

  • Fluorescent Dyes
  • Proteins
  • RNA, Messenger