Optimized RNA ISH, RNA FISH and protein-RNA double labeling (IF/FISH) in Drosophila ovaries

Nat Protoc. 2013 Nov;8(11):2158-79. doi: 10.1038/nprot.2013.136. Epub 2013 Oct 10.


In situ hybridization (ISH) is a powerful technique for detecting nucleic acids in cells and tissues. Here we describe three ISH procedures that are optimized for Drosophila ovaries: whole-mount, digoxigenin-labeled RNA ISH; RNA fluorescent ISH (FISH); and protein immunofluorescence (IF)-RNA FISH double labeling (IF/FISH). Each procedure balances conflicting requirements for permeabilization, fixation and preservation of antigenicity to detect RNA and protein expression with high resolution and sensitivity. The ISH protocol uses alkaline phosphatase-conjugated digoxigenin antibodies followed by a color reaction, whereas FISH detection involves tyramide signal amplification (TSA). To simultaneously preserve antigens for protein detection and enable RNA probe penetration for IF/FISH, we perform IF before FISH and use xylenes and detergents to permeabilize the tissue rather than proteinase K, which can damage the antigens. ISH and FISH take 3 d to perform, whereas IF/FISH takes 5 d. Probe generation takes 1 or 2 d to perform.

Publication types

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

MeSH terms

  • Animals
  • Detergents / pharmacology
  • Drosophila / genetics
  • Drosophila / metabolism*
  • Female
  • In Situ Hybridization / methods*
  • In Situ Hybridization, Fluorescence / methods
  • Ovary / drug effects
  • Ovary / metabolism*
  • RNA / analysis
  • RNA / metabolism
  • RNA-Binding Proteins / analysis
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism
  • Xylenes / pharmacology


  • Detergents
  • RNA-Binding Proteins
  • Xylenes
  • RNA