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 washes. The solutions and buffers required for all steps involved in this method can be found at the end of this protocol.
1. Tissue Preparation and Sectioning
<ol>
 <li>Decapitate and extract subject's brain rapidly, and place it into a plastic mold of adequate size.</li>
 <li>Cover brain with Tissue-Tek embedding medium and rapidly place the plastic mold in a dry-ice/alcohol bath for rapid freezing. Frozen tissue can be stored at -80&deg;C until use.</li>
 <li>Using a cryostat, collect 2 or 3 brain sections per slide onto charged Superfrost Plus slides. Thickness of the sections should be 10-12 &mu;m. Slides can be stored at -80&deg;C until use.</li>
</ol>
2. Preparation of Sephadex G50 Columns for Probe Purification
"Sephadex columns can be purchased from commercial sources, however, we provide below a low-cost alternative for the generation of columns, which will be required for probe purification.
<ol>
 <li>Hydrate adequate amount of Sephadex G50 powder with RNase-free, DEPC-treated water (for example, 2 g of powder in 100 ml of DEPC-treated water), mix solution briefly and store at room temperature to precipitate excess Sephadex G50.</li>
 <li>Remove supernatant (upper layer of water) following Sephadex G50 precipitation.</li>
 <li>Repeat the process above 3- 5 times.</li>
 <li>After the final wash, re-suspend the Sephadex G50 solution in TE buffer (1:1 ratio), and store at 4&deg;C until use.</li>
 <li>Place autoclaved glass wool into a sterile 1 ml syringe and compress it with the plunger to make a compact layer at the bottom, and then place the syringe into a 15 ml Falcon tube.</li>
 <li>Mix well the solution of Sephadex G50 in TE. Fill the syringe/column with this solution.</li>
 <li>Centrifuge the column for 30 sec at 1000 rpm.</li>
 <li>Repeat this procedure until the column is almost completely filled with Sephadex G50 beads.</li>
 <li>Apply 200 &mu;l of column washing buffer to the column and centrifuge it for 2 min at 1000 rpm. Discard flow-through.</li>
 <li>Apply 200 &mu;l of column blocking buffer to the column and spin it for 2 min at 1000 rpm. Repeat this step 4-5 times to equilibrate column. Columns can be stored at 4&deg;C until use if sealed with Parafilm.</li>
</ol>
3. Labeling and Purification of Riboprobes
Below we detail the generation and purification of a single riboprobe. For dFISH, the preparation of each probe will involve the same methodology, except that one of the probes will be labeled with digoxigenin (DIG)-tagged UTP whereas the other with biotin-tagged UTP.
<ol>
 <li>Prepare a concentrated ( > 150 ng/&mu;l) and purified linearized cDNA solution of interest to generate either sense (control) or antisense riboprobes.</li>
 <li>In a 1.5 ml microfuge tube, add 0.5-1 &mu;g of purified cDNA template, 2 &mu;l of 5X probe labeling buffer, 1 &mu;l of 10X DIG (or biotin)-labeling mix, 0.5 &mu;l of RNasin and 1 &mu;l of the appropriate RNA polymerase, and bring the final volume of the solution to 10 &mu;l with RNAse-free (DEPC-treated) water.</li>
 <li>Incubate the solution in a water bath at 37&deg;C for 2 hr.</li>
 <li>Add 1 &mu;l of tRNA (stock; 20 &mu;g/&mu;l), and 39 &mu;l of column blocking buffer to the solution.</li>
 <li>Prepare the Sephadex G50 column for probe purification. To this end, add 50 &mu;l blocking buffer to the column and spin it for 10 sec at 1000 rpm. 
 Repeat this step 2-3 times to equilibrate the column (i.e., the full 50 &mu;l applied to the column are recovered after cycle of centrifugation).</li>
 <li>Apply the probe solution to the Sephadex G50 column, position a new microfuge tube at the bottom of the column, and spin it for 3 min at 1000 rpm to obtain the purified 50 &mu;l of riboprobe solution.</li>
 <li>Assess quality and yield of labeled riboprobe using either a standard formaldehyde-agarose RNA gel, or a spectrophotometer.</li>
</ol>
4. Post-fixation, Acetylation and Hybridization
<ol>
 <li>Remove sections from the -80&deg;C freezer and allow for a room temperature to equilibrate. Subsequently, incubate sections in a cold, freshly-made 3% paraformaldehyde solution for 5 min.</li>
 <li>Briefly rinse sections in phosphate buffered saline (PBS) twice.</li>
 <li>Dehydrate sections through a standard alcohol series (70, 95, and 100%; 2 min each), and let them air-dry.</li>
 <li>Incubate sections in an acetylation solution for 10 min.</li>
 <li>Rinse sections 3 times in 2X SSPE.</li>
 <li>Dehydrate sections once again through the standard alcohol series described above, and allow them to air-dry.</li>
 <li>Prepare an adequate volume of hybridization solution and add both riboprobes to the solution (probe concentration: 1 ng/&mu;l for each probe). Total volume of the hybridization solution is determined based on the number of sections to be hybridized (16 &mu;l of hybridization solution per section).</li>
 <li>Apply adequate volume of the hybridization solution to tissue and coverslip slides ensuring that no air bubbles overlay the tissue.</li>
 <li>Place slides in a metal slide holder. Immerse holder in a mineral oil bath set at 65&deg;C overnight. Ensure that coverslips are facing upward (i.e., the lateral aspect of the slide holder should be in contact with the bottom of the mineral oil container).</li>
</ol>
5. Post-Hybridization Washes
<ol>
 <li>The following day, carefully remove the slide holder from the mineral oil bath and briefly rinse it in chloroform to remove excess oil from the slides.</li>
 <li>Place slides in a 2X SSPE solution for 5-10 min. Coverslips should detach from slides while in solution.</li>
 <li>Transfer the slides to a new 2X SSPE solution, and keep them for 1 hr at room temperature.</li>
 <li>Transfer sections into a solution containing 2X SSPE plus 50% formamide. The temperature of this solution should match the temperature used in the overnight hybridization procedure. Keep slides in this solution for 1.5 hr.</li>
 <li>Transfer sections to a 0.1X SSPE solution pre-warmed to the same temperature of the hybridization step. Incubate sections in this solution for 30 min. Repeat this step for an additional 30 min.</li>
</ol>
6. Detection and Visualization of Riboprobes
<ol>
 <li>Transfer slides to TNT buffer to which 0.3% hydrogen peroxide has been added, for 10 min.</li>
 <li>Wash sections in TNT buffer, 3 times (10 min each).</li>
 <li>Using a DAKO pen, draw a well around the area containing the brain sections. Importantly, caution should be exercised to ensure that sections do not dry.</li>
 <li>Apply 150 &mu;l of TNB buffer to each slide, and incubate sections in this solution for 30 min, in a humid chamber.</li>
 <li>Remove excess TNB solution by tilting slides.</li>
 <li>Apply 150 &mu;l of TNB solution containing peroxidase-conjugated anti-DIG antibody, and store slides in a humid chamber for 2 hr. Antibody concentration should be determined individually for each transcript of interest prior to carrying out dFISH.</li>
 <li>Wash sections in TNT buffer; 3 times, 10 min each.</li>
 <li>Apply 150 &mu;l of Alexa 594-conjugated tyramide working solution to each slide, and store them in a humid chamber for 1 hr. This solution should be prepared according to the manufacturer's directions.</li>
 <li>Wash sections in TNT buffer; 3 times, 10 min each.</li>
 <li>Incubate sections in TNT buffer to which 0.3% hydrogen peroxide has been added for 10- 30 min.</li>
 <li>Wash sections in TNT buffer; 3 times, 10 min each.</li>
 <li>Apply 150 &mu;l of TNB buffer per slide, and keep them in a humid chamber for 30 min.</li>
 <li>Remove excess TNB solution by tilting slides.</li>
 <li>Add 150 &mu;l of TNB solution containing peroxidase-conjugated anti-biotin antibody, and store slides in a humid chamber for 2 hr.</li>
 <li>Wash sections in TNT buffer; 3 times, 10 min each.</li>
 <li>Apply 150 &mu;l of an Alexa 488-conjugated tyramide working solution to the slides and incubate sections for 1 hr. This solution should be prepared according to the manufacturer's recommendations.</li>
 <li>Wash sections in TNT buffer; 3 times, 10 min each.</li>
 <li>Add 150 &mu;l of Hoechst solution (1:1000 in TNT buffer) to the sections and keep them in the humid chamber for 2 min.</li>
 <li>Wash the sections in TNT buffer; 3 times, 5 min each.</li>
 <li>Coverslip sections with a fluorescence-compatible mounting medium (e.g., Vectashield or ProLong antifade).</li>
</ol>
7. Representative Results
<img src="/files/ftp_upload/2102/2102fig1.jpg" alt="Figure 1" /> 
Figure 1. We show here representative dFISH results obtained in the zebra finch brain. Shown are photomicrographs obtained from the caudomedial nidopallium (NCM), the songbird analogue of the mammalian auditory cortex. Brain sections were hybridized with a biotinylated riboprobe directed against parvalbumin (A), a marker for a sub-population of inhibitory neurons, and a DIG-labeled riboprobe directed against the activity-dependent gene zenk (B), a reliable marker for song-driven neurons. C) Overlay of (A) and (B) shows a population of inhibitory neurons that are activated by auditory experience. Arrows and arrowheads indicate cells labeled exclusively with each of the two riboprobes, and asterisks show representative neurons co-expressing both transcripts of interest. Scale bar = 25 &mu;m.

<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

16.0K Views.  University of Rochester. 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.

Video: Double Fluorescence in situ Hybridization in Fresh Brain Sections

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

A combination of different techniques to maximize data collection from mouse tissue is presented.

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A &#34;neurochemical signature&#34; to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.
Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.
Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within ...

A Rat Model of EcoHIV Brain Infection

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. Establishment of the EcoHIV infected rat model for studies of drug abuse and neurocognitive disorders, would be beneficial in the study of neuroHIV and HIV-1 associated neurocognitive disorders (HAND). In the present study, we demonstrate the successful creation of a rat model of active HIV infection using chimeric HIV (EcoHIV). First, the lentiviral construct of EcoHIV was packaged in cultured 293 FT cells for 48 hours. Then, the conditional medium was concentrated and titered. Next, we performed bilateral stereotaxic injections of the EcoHIV-EGFP into F344/N rat brain tissue. One week after infection, EGFP fluorescence signals were detected in the infected brain tissue, indicating that EcoHIV successfully induces an active HIV infection in rats. In addition, immunostaining for the microglial cell marker, Iba1, was performed. The results indicated that microglia were the predominant cell type harboring EcoHIV. Furthermore, EcoHIV rats exhibited alterations in temporal processing, a potential underlying neurobehavioral mechanism of HAND as well as synaptic dysfunction eight weeks after infection. Collectively, the present study extends the EcoHIV model of HIV-1 infection to the rat offering a valuable biological system to study HIV-1 viral reservoirs in the brain as well as HAND and associated comorbidities such as drug abuse.

Here, we present a protocol to establish a new rat model of active HIV infection using chimeric HIV (EcoHIV), which is critical for enhancing our understanding of HIV-1 viral reservoirs in the brain and offering a system to study HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse).

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. ...

Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular characterization. Fluorescence-activated nuclei sorting (FANS) can be used to successfully isolate and study human neuronal nuclei (NeuN+) populations from frozen archival tissue, thereby avoiding problems associated with handling&#160;fresh tissue. However, efforts to similarly isolate astroglia from the non-neuronal (NeuN-) element are lacking. A recently developed and validated immunotagging strategy uses three transcription factor antibodies to simultaneously isolate enriched neuronal (NeuN+), astrocyte (paired box protein 6 (PAX6)+NeuN-), and oligodendrocyte progenitor (OLIG2+NeuN-) nuclei populations from non-diseased, fresh (unfixed) snap-frozen&#160;postmortem human temporal neocortex tissue.
This technique was shown to be useful for the characterization of cell type-specific transcriptome alterations in primary pathological epilepsy neocortex. Transcriptomic analyses confirmed that PAX6+NeuN- sorted populations are robustly enriched for pan-astrocyte markers and capture astrocytes in both resting and reactive conditions. This paper describes the FANS methodology for the isolation of astrocyte-enriched nuclei populations from fresh-frozen human cortex, including tissue dissociation into single-nucleus (sn) suspension; immunotagging of nuclei with anti-NeuN and anti-PAX6 fluorescently conjugated antibodies; FANS gating strategies and quality control metrics for optimizing sensitivity and specificity during sorting and for confirming astrocyte enrichment; and recommended procurement for downstream transcriptome and chromatin accessibility sequencing at bulk or sn resolution. This protocol is applicable for non-necrotic, fresh-frozen, human cortical specimens with various pathologies and recommended postmortem tissue collection within 24 h.

We have developed a method that enriches for and isolates human astrocyte populations from fresh-frozen tissue for use in downstream molecular analyses.