Confocal images were obtained at the University of Iowa Carver Center for Imaging. Funding was provided by an SBIR grant (MH079805) to B.F. Additional support for breeding the mice for this project was provided by NIDCD (5R01DC00559007) to B.F.

<ol>
	<li>Jensen-Smith, H., Gray, B., Muirhead, K., Ohlsson-Wilhelm, B., Fritzsch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-distance+three-color+neuronal+tracing+in+fixed+tissue+using+NeuroVue+dyes.">Long-distance three-color neuronal tracing in fixed tissue using NeuroVue dyes.</a> Immunol Invest. 36, 763-789 (2007).</li><li>Tonniges, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photo-+and+bio-physical+characterization+of+novel+violet+and+near-infrared+lipophilic+fluorophores+for+neuronal+tracing.">Photo- and bio-physical characterization of novel violet and near-infrared lipophilic fluorophores for neuronal tracing.</a> Journal of Microscopy. , (2010).</li><li>Bartheld, C. S. v. o. n., Cunningham, D. E., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+tracing+with+DiI:+decalcification,+cryosectioning,+and+photoconversion+for+light+and+electron+microscopic+analysis.">Neuronal tracing with DiI: decalcification, cryosectioning, and photoconversion for light and electron microscopic analysis.</a> J Histochem Cytochem. 38, 725-733 (1990).</li><li>Gurung, B., Fritzsch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+embryonic+midbrain+thalamic+and+thalamic+auditory+connection+development+in+mice+as+revealed+by+carbocyannine+dye+injection.">Time course of embryonic midbrain thalamic and thalamic auditory connection development in mice as revealed by carbocyannine dye injection.</a> J Comp Neurol. 479, 309-327 (2004).</li><li>Fritzsch, B., Nichols, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DiI+reveals+a+prenatal+arrival+of+efferents+at+the+differentiating+otocyst+of+mice.">DiI reveals a prenatal arrival of efferents at the differentiating otocyst of mice.</a> Hear Res. 65, 51-60 (1993).</li><li>Jahan, I., Kersigo, J., Pan, N., Fritzsch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurod1+suppresses+hair+cell+differentiation+in+ear+ganglia+and+regulates+hair+cell+subtype+development+in+the+cochlea.">Neurod1 suppresses hair cell differentiation in ear ganglia and regulates hair cell subtype development in the cochlea.</a> PloS ONE. 5, e11661-e11661 (2010).</li></ol>

Mouse tissue was collected in accordance with the guidelines and regulations set forth by the University of Iowa Institutional Animal Care and Use Committee (ACURF 0804066).

In this video, we demonstrate a method to combine four techniques in order to maximize data collection and cohesiveness by correlating data within a given animal (figure 3). This approach will reduce the amount of breeding and thus time needed to obtain publishable data while improving information about co-localization and correlative effects of mutants. Although embryonic mice were utilized in this video, adult mouse as well as other animals such as chicken, xenopus, and zebrafish can be analyzed using these techniques as well. When using other species proteinase k digestion may need to be altered. Here we use different fluorescing NeuroVue dyes to specifically label up to three neuronal populations of interest. The benefit of these dyes is that they can be used in mutant mice, which may be problematic when relying solely on immunohistochemistry that may or may not be affected by the mutations to be analyzed, and they label neurons retrogradely and anterogradely 5. A recent study has also increased the number of neuron populations that can be labeled simultaneously to six 2. After, the lipophilic dye tracing the same tissue of interest can then be analyzed using in situ hybridization, for analysis of gene transcription and can be subsequently analyzed using immunohistochemistry for protein distribution. The latter analysis can be supplemented by detailed histology which can add to the phenotype characterization 6. This multifactorial analysis has the potential to gain new insights through correlative analysis within the same animal that may otherwise be improbable or at least in need of more extensive protocols and mouse breeding.

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		<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>in situ Hybridization:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PC DIG wash + block buffer set</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>1585762</td>
<td>&nbsp;</td>
<tr>
<td>Premix 20x SSC Buffer</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>1666681</td>
<td>&nbsp;</td>
<tr>
<td>Proteinase K 20mg/ml</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AM2546</td>
<td>&nbsp;</td>
<tr>
<td>Diethyl Pyrocorbonate (DEPC) </td>
<td>&nbsp;</td>
<td>RPI</td>
<td>D43060</td>
<td>&nbsp;</td>
<tr>
<td>Formamide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F9037</td>
<td>&nbsp;</td>
<tr>
<td>Dextran Sulfate</td>
<td>&nbsp;</td>
<td>RPI</td>
<td>D20020</td>
<td>&nbsp;</td>
<tr>
<td>BM Purple </td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11442074001</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Dig-AP Fab fragments</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11093274910</td>
<td>&nbsp;</td>
<tr>
<td>RNase A enzyme 10mg/ml</td>
<td>&nbsp;</td>
<td>Fermentas</td>
<td>EN0531</td>
<td>&nbsp;</td>
<tr>
<td>RNase Away</td>
<td>&nbsp;</td>
<td>RPI</td>
<td>7003</td>
<td>&nbsp;</td>
<tr>
<td>Salmon Sperm DNA 10mg/ml</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15632-011</td>
<td>&nbsp;</td>
<tr>
<td>Stericup Filter Unit 0.22&mu;m; 500ml</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>SCGVU05RE</td>
<td>&nbsp;</td>
<tr>
<td>Filter Discs 0.2&mu;m; 25mm</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>6780-2502</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate buffered saline PH 7.4 packets</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-5368</td>
<td>&nbsp;</td>
<tr>
<td>Immunohistochemistry:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Goat Serum</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G9023</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T8532</td>
<td>&nbsp;</td>
<tr>
<td>Hoechst Dye</td>
<td>&nbsp;</td>
<td>Polysciences Inc.</td>
<td>9460</td>
<td>&nbsp;</td>
<tr>
<td>Histology:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Poly/Bed 812 Embedding Media/DMP-30 Kit </td>
<td>&nbsp;</td>
<td>Polysciences Inc. </td>
<td>08792-1</td>
<td>&nbsp;</td>
<tr>
<td>Propylene oxide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>110205</td>
<td>&nbsp;</td>
<tr>
<td>Gluteraldehyde Solution 50%</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>G151</td>
<td>&nbsp;</td>
<tr>
<td>DPX Mountant</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>44581</td>
<td>&nbsp;</td>
<tr>
<td>Rocker</td>
<td>&nbsp;</td>
<td>MidSci</td>
<td>55D1114</td>
<td>&nbsp;</td>
<tr>
<td>Heat Block</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>11-715-125D</td>
<td>&nbsp;</td>
<tr>
<td>Rotator</td>
<td>&nbsp;</td>
<td>Thermo</td>
<td>400110Q</td>
<td>&nbsp;</td>
<tr>
<td>Incubator set to 60&deg;</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>11-690-625D</td>
<td>&nbsp;</td>
<tr>
<td>Dye tracing:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microscissors</td>
<td>&nbsp;</td>
<td>Geuder</td>
<td>G-19775</td>
<td>&nbsp;</td>
<tr>
<td>Fine Forceps</td>
<td>&nbsp;</td>
<td>E.M.S</td>
<td>72680-F</td>
<td>&nbsp;</td>
<tr>
<td>NeuroVue dye: Maroon, Red, Jade</td>
<td>&nbsp;</td>
<td>MTTI</td>
<td>FS-100(1,2,6)</td>
<td>&nbsp;</td>
<tr>
<td>1X phosphate buffered saline (PBS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-5368</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting scope</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>M205 FA</td>
<td>&nbsp;</td>
<tr>
<td>Incubator set to 36&deg;</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>11-690-506DQ</td>
<td>&nbsp;</td>
<tr>
<td>Confocal Microscope</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>LSM 510</td>
<td>&nbsp;</td>
<tr>
<td>Slides</td>
<td>&nbsp;</td>
<td>Surgipath</td>
<td>00240</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips</td>
<td>&nbsp;</td>
<td>Surgipath</td>
<td>00106</td>
<td>&nbsp;</td>
<tr>
<td>RPI</td>
<td>&nbsp;</td>
<td>Glycerol</td>
<td>G22025-0.5</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde (4% and 0.4%)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>T353-500</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology at JoVE.com

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

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

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 ...

combining-lipophilic-dye-situ-hybridization-immunohistochemistry

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JoVE Journal

Neuroscience

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