The authors would like to thank all Jarvis lab members who improved the protocol over the years.

<ol>
	<li>Clayton, D. F., Huecas, M. E., Sinclair-Thompson, E. Y., Nastiuk, K. L., Nottebohm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probes+for+rare+mRNAs+reveal+distributed+cell+subsets+in+canary+brain.">Probes for rare mRNAs reveal distributed cell subsets in canary brain.</a> Neuron. 1, 249-261 (1988).</li><li>Wada, K., Sakaguchi, H., Jarvis, E. D., Hagiwara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+glutamate+receptors+in+avian+neural+pathways+for+learned+vocalization.">Differential expression of glutamate receptors in avian neural pathways for learned vocalization.</a> J. Comp. Neurol. 476, 44-64 (2004).</li><li>Haesler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FoxP2+expression+in+avian+vocal+learners+and+non-learners.">FoxP2 expression in avian vocal learners and non-learners.</a> J. Neurosci. 24, 3164-3175 (2004).</li><li>Jarvis, E. D., Nottebohm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor-driven+gene+expression.">Motor-driven gene expression.</a> Proc. Natl. Acad. Sci. U.S.A. 94, 4097-4102 (1997).</li><li>Holzenberger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+expression+of+insulin-like+growth+factor+II+in+the+songbird+brain.">Selective expression of insulin-like growth factor II in the songbird brain.</a> J. Neurosci. 17, 6974-6987 (1997).</li><li>Mahmood, R., Mason, I. <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+of+radioactive+riboprobes+to+RNA+in+tissue+sections.">In-situ hybridization of radioactive riboprobes to RNA in tissue sections.</a> Methods Mol. Biol. 461, 675-686 (2008).</li><li>Wilkinson, D. G., Nieto, M. 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+messenger+RNA+by+in+situ+hybridization+to+tissue+sections+and+whole+mounts.">Detection of messenger RNA by in situ hybridization to tissue sections and whole mounts.</a> Methods Enzymol. 225, 361-373 (1993).</li><li>Acloque, H., Wilkinson, D. G., Nieto, M. A. <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+analysis+of+chick+embryos+in+whole-mount+and+tissue+sections.">In situ hybridization analysis of chick embryos in whole-mount and tissue sections.</a> Methods Cell Biol. 87, 169-185 (2008).</li><li>Moorman, A. F., Houweling, A. C., de Boer, P. A., Christoffels, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+nonradioactive+detection+of+mRNA+in+tissue+sections:+novel+application+of+the+whole-mount+in+situ+hybridization+protocol.">Sensitive nonradioactive detection of mRNA in tissue sections: novel application of the whole-mount in situ hybridization protocol.</a> J. Histochem. Cytochem. 49, 1-8 (2001).</li><li>Horita, H., Wada, K., Rivas, M. V., Hara, E., Jarvis, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dusp1+immediate+early+gene+is+regulated+by+natural+stimuli+predominantly+in+sensory+input+neurons.">The dusp1 immediate early gene is regulated by natural stimuli predominantly in sensory input neurons.</a> J. Comp. Neurol. 518, 2873-2901 (2010).</li><li>Braissant, O., Wahli, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+in+situ+hybridization+protocol+using+non-radioactively+labelled+probes+to+detect+abundant+and+rare+mRNAs+on+tissue+sections.">A simplified in situ hybridization protocol using non-radioactively labelled probes to detect abundant and rare mRNAs on tissue sections.</a> Biochemica. 1, 10-16 (1998).</li><li>Kubikova, L., Wada, K., Jarvis, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+receptors+in+a+songbird+brain.">Dopamine receptors in a songbird brain.</a> J. Comp. Neurol. 518, 741-769 (2010).</li><li>Wada, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+neuroethological+approach+for+identifying+and+characterizing+a+cascade+of+behaviorally+regulated+genes.">A molecular neuroethological approach for identifying and characterizing a cascade of behaviorally regulated genes.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 15212-15217 (2006).</li><li>Jarvis, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avian+brains+and+a+new+understanding+of+vertebrate+brain+evolution.">Avian brains and a new understanding of vertebrate brain evolution.</a> Nat. Rev. Neurosci. 6, 151-159 (2005).</li><li>Hoke, K. L., Ryan, M. J., Wilczynski, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+cues+shift+functional+connectivity+in+the+hypothalamus.">Social cues shift functional connectivity in the hypothalamus.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 10712-10717 (2005).</li><li>Burmeister, S. S., Jarvis, E. D., Fernald, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+behavioral+and+genomic+responses+to+social+opportunity.">Rapid behavioral and genomic responses to social opportunity.</a> PLoS. Biol. 3, e363 (2005).</li><li>Jarvis, E. D., Schwabl, H., Ribeiro, S., 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=Brain+gene+regulation+by+territorial+singing+behavior+in+freely+ranging+songbirds.">Brain gene regulation by territorial singing behavior in freely ranging songbirds.</a> Neuroreport. 8, 2073-2077 (1997).</li><li>Jarvis, E. D., Scharff, C., Grossman, M. R., Ramos, J. A., Nottebohm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=For+whom+the+bird+sings:+context-dependent+gene+expression.">For whom the bird sings: context-dependent gene expression.</a> Neuron. 21, 775-788 (1998).</li><li>Feenders, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mapping+of+movement-associated+areas+in+the+avian+brain:+a+motor+theory+for+vocal+learning+origin.">Molecular mapping of movement-associated areas in the avian brain: a motor theory for vocal learning origin.</a> PLoS One. 3, e1768 (2008).</li><li>Chen, C. C., Fernald, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distributions+of+two+gonadotropin-releasing+hormone+receptor+types+in+a+cichlid+fish+suggest+functional+specialization.">Distributions of two gonadotropin-releasing hormone receptor types in a cichlid fish suggest functional specialization.</a> J. Comp. Neurol. 495, 314-323 (2006).</li></ol>

We have nothing to disclose.

Radioactive in situ hybridization of mRNA expression is widely used for multiple purposes, including for studying regional tissue organization, cell types, and brain functional activity2-5,10,12-14. The later use is on genes whose mRNA expression in the brain is dependent on increased neural activity, often called activity-dependent genes or immediate early genes. With these uses, our method has been applied across multiple species, including in birds, mammals (e.g. human), fish, and amphibians; in multiple tissues, including brain, skin, and muscle; and multiple ages, including hatchlings/neonates, juveniles, adults, and here in whole embryo sections2,3,5,15-17. The special features of our protocol include: (1) It produces a balance between anatomical specificity and quantitative specificity. To quantify the gene expression on the x-ray film, we take digital pictures of the images (ex: Fig. 1A and 1B), use the Photoshop (Adobe) histogram function to measure the pixel density in the regions of interest and subtract the background levels on the film outside of the tissue but still on the glass slide2,4. To quantify expression at the cellular level, we take images of silver grains over cells under high magnification (40-100X; Fig 4). We then use the threshold and measure functions of Image J by Wayne Rasband at NIH to count the number of silver grains in the image, subtract out the background count in a similar area without cells on the glass slide, divide by the number of cells, to obtain a values of expression per cell.4,18 (2) It can be relatively high throughput, allowing processing of 100-200 slides simultaneously, due to the tight coverslip seal created by the mineral oil bath. Standard in-situ hybridization methods take longer to seal the slides with parafilm, nail polish, and other means, where slides take up a lot of space; (3) It is highly sensitive for low abundance transcripts due to Dextran sulfate and Denhardt's solution in the hybridization buffer2,13; (4) The imaging approach in darkfield yields high contrast images due to taking pictures under darkfield illumination on a dissecting microscope4,5. Additionally, It allows sensitive detection of small changes in gene expression, such as in activity-dependent gene expression to identify specific brain regions activated during perception and production of specific behaviors19. The limitations relative to non-radioactive protocols are that the latter are clearer in the cellular resolution and location of the mRNA within the cell, and working with emulsion is very sensitive to manipulation and light. It is possible to combine our method with other methods, such as non-radioactive in situ hybridization to label mRNA expression of more than one gene in the same tissue10,17,20. It can be combined with immunocytochemistry to label both RNA and protein expression on the same sample in order to co-localize the mRNA with certain cell types10. The protocol modifications necessary for such double labeling experiments are described in the cited references.
In summary, our approach facilitates understanding of the timing and cellular location of gene expression in order to understanding region organization, tissue functional activity, and gene function.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Acetic anhydride</td>
 <td>VWR</td>
 <td>MK242002</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Chloroform</td>
 <td>VWR</td>
 <td>BDH1109</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cresyl violet acetate </td>
 <td>Sigma</td>
 <td>C5042</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cryostat</td>
 <td>Thermo scientific</td>
 <td>Microm HM550</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Deionized formamide</td>
 <td>Sigma</td>
 <td>F9037</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>Promega</td>
 <td>P1171</td>
 <td>100mM</td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Sigma</td>
 <td>ED</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Embedding mold</td>
 <td>VWR</td>
 <td>15160-215</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fisher brand Superfrost plus slide </td>
 <td>Fisher Scientific</td>
 <td>22-034-979</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Formaldehyde</td>
 <td>VWR</td>
 <td>BDH0506-4LP</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Formamide</td>
 <td>Sigma</td>
 <td>F7508</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>GENECLEAN kit</td>
 <td>Q-Bio gene </td>
 <td>1001-200 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Kodak BioMax MR film</td>
 <td>Sigma</td>
 <td>Z350370</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Kodak NTB Emulsion</td>
 <td>Carestream Health</td>
 <td>8895666</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Kodak Professional developer D19</td>
 <td>Kodak</td>
 <td>1462593</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Kodak professional Fixer</td>
 <td>Kodak</td>
 <td>1971746</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&#946;-mercaptoethanol </td>
 <td>Calbiochem</td>
 <td>444203</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mineral oil</td>
 <td>VWR</td>
 <td>IC15169491</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NaOH</td>
 <td>VWR</td>
 <td>SX0600-1</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Paraformaldehyde</td>
 <td>Sigma</td>
 <td>76240</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Poly A</td>
 <td>Invitrogen</td>
 <td>POLYA.GF</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>rATP</td>
 <td>Promega</td>
 <td>P1132</td>
 <td>10mM</td>
 </tr>
 <tr>
 <td>rCTP</td>
 <td>Promega</td>
 <td>P1142</td>
 <td>10mM</td>
 </tr>
 <tr>
 <td>rGTP</td>
 <td>Promega</td>
 <td>P1152</td>
 <td>10mM</td>
 </tr>
 <tr>
 <td>RNasin</td>
 <td>Promega</td>
 <td>N2111</td>
 <td>40Units/&mu;l </td>
 </tr>
 <tr>
 <td>S35 UTP</td>
 <td>PerkinElmer</td>
 <td>NEG039C001MC </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Safety-Solve solution </td>
 <td>Safety Solve Research Products International </td>
 <td>111177 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sodium Acetate</td>
 <td>Sigma</td>
 <td>S7899</td>
 <td>3M</td>
 </tr>
 <tr>
 <td>Sodium phosphate dibasic</td>
 <td>Sigma</td>
 <td>S3264</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sodium phosphate monobasic</td>
 <td>Sigma</td>
 <td>S3139</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>SP6 RNA polymerase</td>
 <td>Promega</td>
 <td>P1085</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Staining metal rack</td>
 <td>Electron Microscopy Sciences</td>
 <td>70312-54 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>T7 RNA polymerase</td>
 <td>Promega</td>
 <td>P2075</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tissue-Tek OCT</td>
 <td>Sakura </td>
 <td>4583</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>5x Transcription buffer</td>
 <td>Promega</td>
 <td>P1181</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Triethanolamine</td>
 <td>VWR</td>
 <td>IC15216391</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tris-HCl (1 M, pH 8.0)</td>
 <td>VWR</td>
 <td>101449-446</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>tRNA</td>
 <td>Roche</td>
 <td>10109509001</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>
Solutions:
<ol>
 <li>Reagents for making of riboprobes: 1.5 &mu;l DNA template (0.2-0.3 &mu;g/&mu;l), 2 &mu;l of 5x optimized transcription buffer, 1 &mu;l of 100mM DTT (comes with Polymerase from Promega, mix well at room temperature), 0.3 &mu;l of RNasin (40 units/&mu;l), 1.5 &mu;l of AGC ribonucleotide mix solution, 3.5 &mu;l of S35 UTP, and 1 &mu;l RNA polymerase. Bring up to 10 &mu;l with nuclease-free water.</li>
 <li>AGC ribonucleotide mix solution: mix equal amounts of 10mM ATP, GTP and CTP together.</li>
 <li>Sodium acetate solution for EtOH precipitation to remove free S35 UTP: 40 &mu;l RNase and DNase free water, 5 &mu;l of 3M Sodium Acetate, and 125 &mu;l of 100% EtOH.</li>
 <li>Hybridization buffer (10ml stock): 5 ml of 100% deionized formamide, 600 &mu;l of 5M NaCl, 1M tris-HCl (pH = 8.0), 240 &mu;l of 0.5M EDTA (pH =8.0), 100 &mu;l of 100x Denhart's solution, 100 &mu;l of 1M DTT, 250 &mu;l of 20mg/ml tRNA, 125 &mu;l of 20 mg/ml poly A, and 1 g of Sodium Dextran Sulfate. Add DEPC-treated water to bring the total volume to 10 ml. Shake vigorously and then incubate at 55&deg;C until all sodium dextran sulfate is dissolved. Store the hybridization buffer in -20&deg;C, which is good for ~6 months. </li>
 <li>4% buffered paraformaldehyde solution: Add 40 g of paraformaldehyde in 760 ml distilled water in a flask designated for paraformaldehyde, heat to 50&deg;C on a hot plate while stirring. Add 320 &mu;l of 10N NaOH to help dissolve the paraformaldehyde. After dissolving (~10 min), add 100 ml of 10x PBS, and bring the volume up with distilled water until 1 liter. Stir and heat the solution until paraformaldehyde is dissolved. The pH should be 7.4.</li>
 <li>Acetylation buffer: 13.6 ml of triethanolamine plus 2.52 ml of acetic anhydride in 1 liter of distilled water.</li>
 <li>20x SSPE solution: 3M NaCl, 200 mM NaH2PO4-H2O, and 200 mM EDTA in distilled water. Adjust solution to pH 7.4 with 10N NaOH. </li>
 <li>10x PBS buffer: 80g of NaCl, 2.0g of KCl, 14.4g of Na2HPO4, and 2.4g of KH2PO4 in distilled water. Adjust solution to pH 7.0 and bring total volume to 1 liter. </li>
 <li>Second wash solution: 0.1% &#946;-mercaptoethanol and 50% formamide in 2x SSPE</li>
 <li>Cresyl violet acetate solution: 3% cresyl violet acetate in tap water (distilled water prevents good staining), dissolved overnight with stirring in a flask at room temperature. Filter with vacuum suction through a 1mm Whatman filter paper and Buchner funnel. </li> 
</ol>

radioactive in situ hybridization for detecting diverse gene expression patterns in tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Watch this Scientific Journal Video about Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue at JoVE.com

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

radioactive-situ-hybridization-for-detecting-diverse-gene-expression

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15.5K Views.  Duke University. This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.