This work was supported by NIH grants R21MH082360 (BGC) and R01HD056315 (NRM) as well as the University of Georgia.

1. Riboprobe generation via in vitro transcription
<ol>
	<li>Preparing PCR products for in vitro transcription templates.
	<ol>
		<li>Designing PCR primers with phage transcription promoter sequences in their 5' ends. 
		Note: The promoter sequence added to 5'end of the sense-strand PCR primer will be used for transcribing the sense probe, and the promoter sequence added to 5'end of the antisense PCR primer will be used for synthesizing the anti-sense probe1-3. We...

<ol>
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A., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exogenous+retinoic+acid+rapidly+induces+anterior+ectopic+expression+of+murine+Hox-2+genes+in+vivo.">Exogenous retinoic acid rapidly induces anterior ectopic expression of murine Hox-2 genes in vivo.</a> Development. 116, 357-368 (1992).</li><li>Krauss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+pax[zf-a]:+a+paired+box-containing+gene+expressed+in+the+neural+tube.">Zebrafish pax[zf-a]: a paired box-containing gene expressed in the neural tube.</a> EMBO J. 10, 3609-3619 (1991).</li><li>Hemmati-Brivanlou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+specific+mRNAs+in+Xenopus+embryos+by+whole-mount+in+situ+hybridization.">Localization of specific mRNAs in Xenopus embryos by whole-mount in situ hybridization.</a> Development. 110, 325-330 (1990).</li><li>Harland, R. 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No conflicts of interest declared.

The methods described in this protocol have been adapted from a number of different sources and optimized for whole mount E8.5-E11.5 day old mouse embryos. Methods for whole mount in situ hybridization of vertebrate embryos first appeared in the early 1990s 2,5-12. This protocol was adapted primarily from methods developed for Xenopus embryos 7,8 as well as mouse 2,11. In our protocol a great deal of emphasis is placed on careful quality assessment of the probe. Careful atten...

<table border="1">
	<tbody>
		<tr>
			<th>Name</th>
			<th>Type</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments</th>
		</tr>
		<tr>
			<td>Digoxigenin-11-uridine-5'-triphosphate</td>
			<td>Reagent</td>
			<td>Roche</td>
			<td>11209256910</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ribonucleoside Triphosphate Set</td>
			<td>Reagent</td>
			<td>Roche</td>
			<td>11277057001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Quick Spin Columns for radiolabeled RNA purification</td>
			<td>Supply</td>
			<td>Roche</td>
			<td>11274015001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>T7 RNA polymerase</td>
			<td>Reagent</td>
			<td>Roche</td>
			<td>10881767001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SP6 RNA polymerase</td>
			<td>Reagent</td>
			<td>Roche</td>
			<td>10810274001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>T3 RNA polymerase</td>
			<td>Reagent</td>
			<td>Roche</td>
			<td>11031163001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CTP, [&alpha;-32P]- 800Ci/mmol 10mCi/ml , 250 &mu;Ci			</td>
			<td>Reagent</td>
			<td>Perkin Elmer</td>
			<td>BLU008X250UC</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DNase I recombinant, RNase-free	</td>
			<td>Reagent</td>
			<td>Roche</td>
			<td>4716728001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Urea,Colorless-to-white Crystals or Crystalline Powder</td>
			<td>Reagent</td>
			<td>Fisher</td>
			<td>BP169-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gel loading buffer</td>
			<td>Reagent</td>
			<td>Ambion</td>
			<td>AM8547</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hybond-N+, Amersham</td>
			<td>Supply</td>
			<td>GE Healthcare</td>
			<td>RPN82B</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>UV Stratalinker 2400</td>
			<td>Equipment</td>
			<td>Stratagene</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Diethyl pyrocarbonate</td>
			<td>Reagent</td>
			<td>Sigma</td>
			<td>D5758-100ML</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heparin sodium</td>
			<td>Reagent</td>
			<td>Acros</td>
			<td>41121-0010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>hydrogen peroxide 30% in water</td>
			<td>Reagent</td>
			<td>Fisher scientific</td>
			<td>BP2633-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gluteraldehyde, 8% EM grade</td>
			<td>Reagent</td>
			<td>Polysciences</td>
			<td>0/710</td>
			<td>Use with caution according to manufacture's instructions</td>
		</tr>
		<tr>
			<td>Blocking reagent</td>
			<td>Reagent</td>
			<td>Roche</td>
			<td>11096176001</td>
			<td>Make into 5% stock and store at -20&deg; C</td>
		</tr>
		<tr>
			<td>Proteinase K			</td>
			<td>Reagent</td>
			<td>Roche</td>
			<td>3115852001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rnase A				 </td>
			<td>Reagent</td>
			<td>Roche</td>
			<td>10109142001</td>
			<td>Make as 10 mg/ml stock and store at -20&deg; C.</td>
		</tr>
		<tr>
			<td>Rnase T1</td>
			<td>Reagent</td>
			<td>Roche</td>
			<td>10109193001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ultrapure Formamide</td>
			<td>Reagent</td>
			<td>Invitrogen</td>
			<td>15515-026</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Levamisole hydrochloride</td>
			<td>Reagent</td>
			<td>ICN Biomedicals. Inc</td>
			<td>155228</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ribonucleic acid from torula yeast,Type VI				</td>
			<td>Reagent</td>
			<td>Sigma</td>
			<td>R6625</td>
			<td>phenol/chloroform extracted several times and precipitated, resuspended in DEPC-dH2O and stored at -20&deg; C</td>
		</tr>
		<tr>
			<td>Anti-Digoxigenin-AP Fab fragments</td>
			<td>Reagent</td>
			<td>Roche</td>
			<td>11093274910</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BM Purple AP Substrate, precipitating. Ready-to-use solution.</td>
			<td>Reagent</td>
			<td>Roche</td>
			<td>11 442 074 001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>4mL, Clear, Closed Top, Storage Vial Convenience Kit</td>
			<td>Supply</td>
			<td>National scientific</td>
			<td>B7800-2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Shake N Bake hybridization oven</td>
			<td>Equipment</td>
			<td>Boekel Scientific</td>
			<td>136400</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biopsy Sure-Tek</td>
			<td>Supply </td>
			<td>Fisherbrand</td>
			<td>15-200-402C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Paraplast Plus tissue embedding medium</td>
			<td>Reagent</td>
			<td>Fisherbrand</td>
			<td>23-021-400</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Peel-A-Way disposable plastic tissue embedding molds</td>
			<td>Supply</td>
			<td>Polysciences</td>
			<td>18646A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Colorfrost Plus Microscope slides</td>
			<td>Supply</td>
			<td>Fisherbrand</td>
			<td>12-550-17</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nuclear Fast Red</td>
			<td>Reagent</td>
			<td>Sigma</td>
			<td>N8002</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cytoseal 60</td>
			<td>Reagent</td>
			<td>Richard-Allan Scientific</td>
			<td>8310-16</td>
			<td>&nbsp;</td>
		</tr>
		
	</tbody>
</table>

whole mount in situ hybridization of e8.5 to e11.5 mouse embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Watch this Scientific Journal Video about Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos at JoVE.com

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

whole-mount-in-situ-hybridization-of-e85-to-e115-mouse-embryos

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25.7K visualizações.  University of Georgia. This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.