FISHing for chick genes: Triple-label whole-mount fluorescence in situ hybridization detects simultaneous and overlapping gene expression in avian embryos

Dev Dyn. 2004 Mar;229(3):651-7. doi: 10.1002/dvdy.20005.


Multi-color whole-mount in situ hybridization is a powerful technique for comparing the spatial expression patterns of two or more genes in developing embryos. We have developed an amplified triple-label whole-mount fluorescence in situ hybridization (FISH) protocol that permits detection of three different mRNAs in a single embryo. Our protocol uses simultaneous in situ hybridization to haptenylated riboprobes, followed by sequential antibody detection using anti-hapten antibodies conjugated to horseradish peroxidase, and the tyramide signal amplification (TSA) fluorescence detection system. Conventional fluorescence microscopy identifies areas of overlapping gene expression at the tissue level, whereas confocal fluorescence microscopy permits single-cell resolution and differentiates specialized cell types within a given tissue. This protocol will provide researchers engaged in the use of FISH with a solid starting point for adapting their own in situ hybridization protocols, either alone or in combination with immunohistochemistry or green fluorescence protein colocalization.

Publication types

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

MeSH terms

  • Animals
  • Chick Embryo
  • DNA-Binding Proteins / metabolism
  • Developmental Biology / methods*
  • Gene Expression Regulation, Developmental*
  • Glycoproteins / metabolism
  • In Situ Hybridization, Fluorescence / methods*
  • Intercellular Signaling Peptides and Proteins / metabolism
  • Mesoderm / metabolism
  • Microscopy, Confocal
  • Microscopy, Fluorescence / methods
  • PAX2 Transcription Factor
  • Time Factors
  • Transcription Factors / metabolism


  • DNA-Binding Proteins
  • Glycoproteins
  • Intercellular Signaling Peptides and Proteins
  • PAX2 Transcription Factor
  • Transcription Factors
  • chordin