Embryonic gene expression resolved at the cellular level by fluorescence in situ hybridization.
Tyramide signal amplification has successfully been applied to enhance detection limits of both immunological reactions and in situ hybridization methods. The technique uses short-range deposition of activated tyramide mediated by horseradish peroxidase. We have adapted this method to fluorescence in situ hybridization on embryonic tissue sections using fluorophore-labeled tyramide. The sensitivity of the procedure was sufficient to analyze the embryonic expression of mRNAs encoding both transcription factors and structural proteins. Combining fluorescence in situ hybridization and immunofluorescence with confocal microscopy allows the simultaneous detection of distinct mRNA species or of mRNAs together with proteins on the cellular level. Thus, the cell types expressing a particular gene at a given developmental stage can be studied even if no antibody to the gene product of interest is available. Moreover, the technique allows to study in situ the combinatorial marker expression that characterizes progenitor stages of a given cell lineage.
Paratore
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C
Institute of Cell Biology, Swiss Federal Institute of Technology, Zürich.
Suter
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U
Sommer
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L
D000818:Animals; D002232:Carbocyanines; D004268:DNA-Binding Proteins / Q000235:genetics; D005314:Embryonic and Fetal Development; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D015870:Gene Expression; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008168:Lung / Q000196:embryology / Q000378:metabolism; D051379:Mice; D012680:Sensitivity and Specificity; D014157:Transcription Factors / Q000235:genetics
D002232:Carbocyanines; D004268:DNA-Binding Proteins; C513242:Etv5 protein, mouse; D005456:Fluorescent Dyes; D014157:Transcription Factors; C089187:cyanine dye 3; C065788:transcription factor PEA3; D016650:Fluorescein-5-isothiocyanate