Simultaneous detection of the subcellular localization of RNAs and proteins in cultured cells by combined multicolor RNA-FISH and IF

Methods. 2017 Apr 15:118-119:101-110. doi: 10.1016/j.ymeth.2016.09.010. Epub 2016 Sep 21.

Abstract

Fluorescence in situ hybridization (FISH) and immunofluorescence (IF) are sensitive techniques used for detecting nucleic acids and proteins in cultured cells. However, these techniques are rarely applied together, and standard protocols are not readily compatible for sequential application on the same specimen. Here, we provide a user-friendly step-by-step protocol to perform multicolor RNA-FISH in combination with IF to simultaneously detect the subcellular localization of distinct RNAs and proteins in cultured cells. We demonstrate the use of our protocol by analyzing changes in the subcellular distribution of RNAs and proteins in cells exposed to a variety of stress conditions.

Keywords: Antibody; FISH; GFP; Immunofluorescence; Nucleic acids; Stress granule.

Publication types

  • Review
  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • Antibodies / chemistry
  • Arsenites / toxicity
  • Aurintricarboxylic Acid / analogs & derivatives
  • Aurintricarboxylic Acid / pharmacology
  • Base Sequence
  • Cytoplasmic Granules / drug effects
  • Cytoplasmic Granules / metabolism*
  • Cytoplasmic Granules / ultrastructure
  • DNA Helicases / genetics*
  • DNA Helicases / metabolism
  • Fluorescent Antibody Technique / methods*
  • Genes, Reporter
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • Hot Temperature
  • Humans
  • Hydrogen Peroxide / pharmacology
  • In Situ Hybridization, Fluorescence / methods*
  • Oligonucleotide Probes / chemistry
  • Oligonucleotide Probes / metabolism
  • Poly-ADP-Ribose Binding Proteins / genetics*
  • Poly-ADP-Ribose Binding Proteins / metabolism
  • Protein Binding
  • RNA Helicases / genetics*
  • RNA Helicases / metabolism
  • RNA Recognition Motif Proteins / genetics*
  • RNA Recognition Motif Proteins / metabolism
  • RNA, Ribosomal, 18S / genetics*
  • RNA, Ribosomal, 18S / metabolism
  • RNA, Ribosomal, 28S / genetics*
  • RNA, Ribosomal, 28S / metabolism
  • Sodium Compounds / toxicity
  • Stress, Physiological
  • Thapsigargin / pharmacology

Substances

  • Antibodies
  • Arsenites
  • Oligonucleotide Probes
  • Poly-ADP-Ribose Binding Proteins
  • RNA Recognition Motif Proteins
  • RNA, Ribosomal, 18S
  • RNA, Ribosomal, 28S
  • Sodium Compounds
  • Green Fluorescent Proteins
  • Aurintricarboxylic Acid
  • sodium arsenite
  • Thapsigargin
  • aurin
  • Hydrogen Peroxide
  • DNA Helicases
  • G3BP1 protein, human
  • RNA Helicases