FISHing for Chick Genes: Triple-Label Whole-Mount Fluorescence in Situ Hybridization Detects Simultaneous and Overlapping Gene Expression in Avian Embryos

Nathaniel Denkers, Pilar García-Villalba, Christopher K. Rodesch, Kandice R. Nielson, Teri Jo Mauch

Research output: Contribution to journalArticle

55 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)651-657
Number of pages7
JournalDevelopmental Dynamics
Volume229
Issue number3
DOIs
StatePublished - Mar 1 2004

Fingerprint

Overlapping Genes
Fluorescence In Situ Hybridization
In Situ Hybridization
Embryonic Structures
Fluorescence Microscopy
Gene Expression
Fluorescence
Genes
Haptens
Horseradish Peroxidase
Confocal Microscopy
Anti-Idiotypic Antibodies
Color
Immunohistochemistry
Research Personnel
Messenger RNA
Antibodies
Proteins

Keywords

  • Avian
  • Chick
  • Chordin
  • Colocalization
  • Confocal
  • Embryo
  • Fluorescence in situ hybridization
  • Mesoderm
  • Paraxis
  • Pax-2
  • Whole-mount

ASJC Scopus subject areas

  • Developmental Biology

Cite this

FISHing for Chick Genes : Triple-Label Whole-Mount Fluorescence in Situ Hybridization Detects Simultaneous and Overlapping Gene Expression in Avian Embryos. / Denkers, Nathaniel; García-Villalba, Pilar; Rodesch, Christopher K.; Nielson, Kandice R.; Mauch, Teri Jo.

In: Developmental Dynamics, Vol. 229, No. 3, 01.03.2004, p. 651-657.

Research output: Contribution to journalArticle

Denkers, Nathaniel ; García-Villalba, Pilar ; Rodesch, Christopher K. ; Nielson, Kandice R. ; Mauch, Teri Jo. / FISHing for Chick Genes : Triple-Label Whole-Mount Fluorescence in Situ Hybridization Detects Simultaneous and Overlapping Gene Expression in Avian Embryos. In: Developmental Dynamics. 2004 ; Vol. 229, No. 3. pp. 651-657.
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