Work supported by grants of NIH/NIDCD and the Schmitt Foundation to RP.

This protocol was developed and refined based on standard radioactive and non-radioactive in-situ hybridization methods, previously developed by us and others to detect one or two transcript species in brain tissue 1-7. The protocol described below has a total length of 2 or 3 days, depending on the number of procedural interruptions chosen by the end user. All steps detailed below are to be conducted at room temperature, with the exception of the riboprobe hybridization step and post-hybridization wa...

<ol>
	<li>Jin, L., Lloyd, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization:+methods+and+applications.">In situ hybridization: methods and applications.</a> J Clin Lab Anal. 11, 2-9 (1997).</li><li>Stoler, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization.">In situ hybridization.</a> Clin Lab Med. 10, 215-236 (1990).</li><li>Komminoth, P., Werner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+and+signal+amplification:+approaches+to+increase+the+sensitivity+of+in+situ+hybridization.">Target and signal amplification: approaches to increase the sensitivity of in situ hybridization.</a> Histochem Cell Biol. 108, 325-3233 (1997).</li><li>Kessler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+digoxigenin:anti-digoxigenin+(DIG)+technology--a+survey+on+the+concept+and+realization+of+a+novel+bioanalytical+indicator+system.">The digoxigenin:anti-digoxigenin (DIG) technology--a survey on the concept and realization of a novel bioanalytical indicator system.</a> Mol Cell Probes. 5, 161-205 (1991).</li><li>Panoskaltsis-Mortari, A., Bucy, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+with+digoxigenin-labeled+RNA+probes:+facts+and+artifacts.">In situ hybridization with digoxigenin-labeled RNA probes: facts and artifacts.</a> Biotechniques. 18, 300-307 (1995).</li><li>Qian, X., Lloyd, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+signal+amplification+methods+for+in+situ+hybridization.">Recent developments in signal amplification methods for in situ hybridization.</a> Diagn Mol Pathol. 12, 1-13 (2003).</li><li>Mello, C. V., Jarvis, E. D., Denisenko, N., Rivas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+song-regulated+genes+in+the+brain+of+songbirds.">Isolation of song-regulated genes in the brain of songbirds.</a> Methods Mol Biol. 85, 205-217 (1997).</li><li>Pinaud, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+neurons+participate+in+the+brain's+response+to+birdsong+auditory+stimulation.">GABAergic neurons participate in the brain's response to birdsong auditory stimulation.</a> Eur J Neurosci. 20, 1318-1330 (2004).</li><li>Velho, T. A., Pinaud, R., Rodrigues, P. V., Mello, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-induction+of+activity-dependent+genes+in+songbirds.">Co-induction of activity-dependent genes in songbirds.</a> Eur J Neurosci. 22, 1667-1678 (2005).</li><li>Tremere, L. A., Jeong, J. K., Pinaud, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estradiol+shapes+auditory+processing+in+the+adult+brain+by+regulating+inhibitory+transmission+and+plasticity-associated+gene+expression.">Estradiol shapes auditory processing in the adult brain by regulating inhibitory transmission and plasticity-associated gene expression.</a> J Neurosci. 29, 5949-5963 (2009).</li><li>Pinaud, R., Mello, C. V., Velho, T. A., Wynne, R. D., Tremere, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+two+mRNA+species+at+single-cell+resolution+by+double-fluorescence+in+situ+hybridization.">Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization.</a> Nat Protoc. 3, 1370-1379 (2008).</li><li>Pinaud, R., Jeong, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duplex+fluorescence+in+situ+hybridization+in+the+study+of+gene+co-regulation+in+the+vertebrate+brain.+Methods+Mol+Biol.">Duplex fluorescence in situ hybridization in the study of gene co-regulation in the vertebrate brain. Methods Mol Biol.</a> 611, 115-129 (2010).</li></ol>

No conflicts of interest declared.

We have used this protocol to study how the vertebrate brain is neurochemically and functionally organized, and to determine how behaviorally-relevant sensory stimuli impact the genomic machinery of neurons in the adult brain 8-10. We have successfully used this method in brain tissue from mice, rats and songbirds, but anticipate that this protocol will be easily adaptable to brain sections obtained from an array of vertebrate species and, perhaps, non-neural tissues. Key controls required to obtaining reliabl...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DEPC</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>IC15090225</td>
<td>toxic substance</td>
</tr>
<tr>
<td>PCR purification kit</td>
<td>&nbsp;</td>
<td>QIAgen</td>
<td>28104</td>
<td>&nbsp;</td>
<tr>
<td>Formaldehyde</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>BDH0506-4LP
</td>
<td>toxic substance</td>
</tr>
<tr>
<td>T3 RNA polymerase</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11031163001</td>
<td>&nbsp;</td>
<tr>
<td>T7 RNA polymerase</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>10881767001</td>
<td>&nbsp;</td>
<tr>
<td>Tris-HCl (1 M, pH 7.5)</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>IC816124</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2 (1 M)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>449172</td>
<td>&nbsp;</td>
<tr>
<td>Spermidine (1 M)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S0266</td>
<td>&nbsp;</td>
<tr>
<td>DIG RNA labeling mix</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>NC9380805</td>
<td>&nbsp;</td>
<tr>
<td>Biotin RNA labeling mix</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>NC9440104</td>
<td>&nbsp;</td>
<tr>
<td>RNasin</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>N2111</td>
<td>&nbsp;</td>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>05491</td>
<td>&nbsp;</td>
<tr>
<td>DTT</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D5545</td>
<td>&nbsp;</td>
<tr>
<td>Sephadex G50</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>95016-772</td>
<td>&nbsp;</td>
<tr>
<td>EDTA</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>101384-758</td>
<td>&nbsp;</td>
<tr>
<td>SDS</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L4390</td>
<td>&nbsp;</td>
<tr>
<td>Transfer (t)RNA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15401011</td>
<td>&nbsp;</td>
<tr>
<td>10X MOPS</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>14221-398</td>
<td>toxic substance</td>
</tr>
<tr>
<td>Paraformaldehyde</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>AAAL04737-36</td>
<td>toxic substance</td>
</tr>
<tr>
<td>Sodium phosphate monobasic</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S3139</td>
<td>&nbsp;</td>
<tr>
<td>Sodium phosphate dibasic</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S3264</td>
<td>&nbsp;</td>
<tr>
<td>NaOH</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>SX0600-1</td>
<td>&nbsp;</td>
<tr>
<td>Triethanolamine</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>IC15216391</td>
<td>&nbsp;</td>
<tr>
<td>Acetic anhydride</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>MK242002</td>
<td>toxic substance</td>
</tr>
<tr>
<td>SSPE</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>82021-488</td>
<td>&nbsp;</td>
<tr>
<td>Formamide</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>JT4028-1</td>
<td>toxic substance</td>
</tr>
<tr>
<td>Poly A</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>POLYA.GF</td>
<td>&nbsp;</td>
<tr>
<td>Mineral oil</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>IC15169491</td>
<td>&nbsp;</td>
<tr>
<td>Chloroform</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>BDH1109</td>
<td>organic solvent</td>
</tr>
<tr>
<td>Deionized formamide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F9037</td>
<td>toxic substance</td>
</tr>
<tr>
<td>Hydrogen peroxide</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>VW36901</td>
<td>toxic substance</td>
</tr>
<tr>
<td>Tween 20</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1379</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>J. T. Baker</td>
<td>X198-05</td>
<td>&nbsp;</td>
<tr>
<td>Anti-DIG HRP</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11093274910</td>
<td>&nbsp;</td>
<tr>
<td>Anti-biotin peroxidase</td>
<td>&nbsp;</td>
<td>Vector</td>
<td>SP3010</td>
<td>&nbsp;</td>
<tr>
<td>TSA Alexa Fluor 594</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>T20935</td>
<td>&nbsp;</td>
<tr>
<td>TSA Alexa Fluor 488</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>T20932</td>
<td>&nbsp;</td>
<tr>
<td>Hoechst</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>200025-538</td>
<td>&nbsp;</td>
<tr>
<td>Vectashield</td>
<td>&nbsp;</td>
<td>Vector</td>
<td>H-1000</td>
<td>&nbsp;</td>
<tr>
<td>embedding mold</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>15160-157</td>
<td>&nbsp;</td>
<tr>
<td>cryostat</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>CM 1850</td>
<td>&nbsp;</td>
<tr>
<td>Superfrost plus slide</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89033-052</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
			Solutions
<ol>
	<li>Column washing buffer: 10 mM Tris-HCl, 0.15 M NaCl, 0.05 mM EDTA, 50 &mu;g/&mu;l tRNA, 0.1% SDS in 50 ml DEPC-treated water.</li>
	<li>Column blocking buffer: 10 mM Tris-HCl, 50 mM NaCl, 0.1 mM EDTA in 50 ml DEPC-treated water.</li>
	<li>TNT buffer: 60 ml of 1 M Tris-HCl, 18 ml of 5 M NaCl and 1.8 ml of Triton X-100 in 600 ml of DEPC-treated water.</li>
	<li>TNB buffer: 100 mM Tris-HCl, 8.3 &mu;g/&mu;l BSA, 0.15 M NaCl and 3% Triton X-100 in DEPC-treated water.</li>
	<li>TE buffer: 10 mM Tris-HCl, pH 7.5 plus 1 mM EDTA, pH 7.5.</li>
	<li>Acetylation solution: 2.7 ml of triethanolamine plus 0.5 ml of acetic anhydride in 200 ml of DEPC-treated water.</li>
	<li>Hybridization solution: 50% formamide, 2X SSPE, 2 &mu;g/&mu;l tRNA, 1 &mu;g/&mu;l BSA and 1 &mu;g/&mu;l poly A in DEPC-treated water. It needs 16 l per section.</li>
</ol>		</div>

double fluorescence in situ hybridization in fresh brain sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Watch this Scientific Journal Video about Double Fluorescence in situ Hybridization in Fresh Brain Sections at JoVE.com

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

double-fluorescence-in-situ-hybridization-in-fresh-brain-sections

Research

JoVE Journal

Neuroscience

15.9K visualizações.  University of Rochester. Este protocolo envolve um não-radioativo In-situ Processo de hibridação que permite a identificação simultânea de duas espécies de transcrição, com uma resolução única célula, em seções finas do cérebro dos vertebrados.