Evol Ecol Res 17: 603-617 (2016)     Full PDF if your library subscribes.

A fluorescence in situ hybridization (FISH) protocol
for stickleback tissue

Noelle James1, Xiaochen Liu2 and Alison Bell1,3

1Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA,  2Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign,  Urbana, Illinois, USA and  3School of Integrative Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

Correspondence: N. James, Neuroscience Program, University of Illinois at Urbana-Champaign, 505 South Goodwin Avenue, Urbana, IL 61801, USA. email: nholmes3@life.illinois.edu


Background: Threespine stickleback are an important model for behaviour and evolutionary studies. A growing number of quantitative trait loci (QTL) and gene expression studies are identifying genes related to ecologically important traits in sticklebacks. In order to visualize the expression of candidate genes, we developed a fluorescence in situ hybridization (FISH) protocol.

Methods: We present a protocol for FISH on fresh or flash-frozen dissected tissue, using either cryo- or paraffin embedding. The protocol covers probe design guidelines and synthesis, sample embedding, sectioning, and the hybridization process. The protocol is optimized for brain tissue. Key steps for modifying the protocol for other tissues are noted.

Results: The FISH protocol resulted in specific labelling under all combinations of dissection and embedding conditions. Paraffin embedding preserved morphology better than cryo-embedding. We provide representative results showing the expression of glial fibrillary acidic protein (GFAP ), oxytocin receptor (OXTR), and tyrosine hydroxylase (TH ) in the brain.

Keywords: fluorescence in situ hybridization, fluorescence methods, Gasterosteus aculeatus, gene expression, gene localization, in situ hybridization, RNA, threespine stickleback.

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