TY - JOUR
T1 - A fluorescence in situ hybridization (FISH) protocol for stickleback tissue
AU - James, Noelle
AU - Liu, Xiaochen
AU - Bell, Alison
N1 - Funding Information:
We thank Lisa Stubbs and Chris Seward for generous use of equipment and advice. Most imaging was done on the Zeiss Axiovert 200M from the Core Facilities at the Carl R. Woese Institute for Genomic Biology. Funding was provided by NSF (IOS 1121980), NIH (R01 GM082937), UIUC and the Simons Foundation.
Publisher Copyright:
© 2016 Noelle James.
PY - 2016/7
Y1 - 2016/7
N2 - 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 cryoembedding. We provide representative results showing the expression of glial fibrillary acidic protein (GFAP), oxytocin receptor (OXTR), and tyrosine hydroxylase (TH) in the brain.
AB - 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 cryoembedding. We provide representative results showing the expression of glial fibrillary acidic protein (GFAP), oxytocin receptor (OXTR), and tyrosine hydroxylase (TH) in the brain.
KW - Fluorescence in situ hybridization
KW - Fluorescence methods
KW - Gasterosteus aculeatus
KW - Gene expression
KW - Gene localization
KW - In situ hybridization
KW - RNA
KW - Threespine stickleback
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M3 - Article
AN - SCOPUS:84978656608
SN - 1522-0613
VL - 17
SP - 603
EP - 617
JO - Evolutionary Ecology Research
JF - Evolutionary Ecology Research
IS - 4
ER -