Name: whole mount in situ hybridization of e8.5 to e11.5 mouse embryos
Uploaded: 2011-10-10T12:00:00
Description: Watch this Scientific Journal Video about Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos at JoVE.com

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>
	<li>Divjak, M., Glare, E. M., Walters, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+non-radioactive+in+situ+hybridization+in+human+airway+tissues:+use+of+PCR-generated+templates+for+synthesis+of+probes+and+an+antibody+sandwich+technique+for+detection+of+hybridization.">Improvement of non-radioactive in situ hybridization in human airway tissues: use of PCR-generated templates for synthesis of probes and an antibody sandwich technique for detection of hybridization.</a> J. Histochem. Cytochem. 50, 541-548 (2002).</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>Logel, J., Dill, D., Leonard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+cRNA+probes+from+PCR-generated+DNA.">Synthesis of cRNA probes from PCR-generated DNA.</a> Biotechniques. 13, 604-610 (1992).</li><li>Maddox, D. M., Condie, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+expression+of+a+glutamate+decarboxylase+gene+in+multiple+non-neural+tissues+during+mouse+development.">Dynamic expression of a glutamate decarboxylase gene in multiple non-neural tissues during mouse development.</a> BMC Dev Biol. 1, 1-1 (2001).</li><li>Conlon, R. 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. M. <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:+an+improved+whole-mount+method+for+Xenopus+embryos.">In situ hybridization: an improved whole-mount method for Xenopus embryos.</a> Methods Cell Biol. 36, 685-695 (1991).</li><li>Herrmann, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+pattern+of+the+Brachyury+gene+in+whole-mount+TWis/TWis+mutant+embryos.">Expression pattern of the Brachyury gene in whole-mount TWis/TWis mutant embryos.</a> Development. 113, 913-917 (1991).</li><li>Conlon, R. A., Herrmann, B. G. <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+postimplantation+embryo+whole+mounts.">Detection of messenger RNA by in situ hybridization to postimplantation embryo whole mounts.</a> Methods Enzymol. 225, 373-383 (1993).</li><li>Parr, B. A., Shea, M. J., Vassileva, G., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Wnt+genes+exhibit+discrete+domains+of+expression+in+the+early+embryonic+CNS+and+limb+buds.">Mouse Wnt genes exhibit discrete domains of expression in the early embryonic CNS and limb buds.</a> Development. 119, 247-261 (1993).</li><li>Rosen, B., Beddington, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-mount+in+situ+hybridization+in+the+mouse+embryo:+gene+expression+in+three+dimensions.">Whole-mount in situ hybridization in the mouse embryo: gene expression in three dimensions.</a> Trends Genet. 9, 162-167 (1993).</li><li>Quiring, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+expression+screening+by+automated+whole-mount+in+situ+hybridization.">Large-scale expression screening by automated whole-mount in situ hybridization.</a> Mech. Dev. 121, 971-976 (2004).</li><li>Thut, C. J., Rountree, R. B., Hwa, M., Kingsley, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+large-scale+in+situ+screen+provides+molecular+evidence+for+the+induction+of+eye+anterior+segment+structures+by+the+developing+lens.">A large-scale in situ screen provides molecular evidence for the induction of eye anterior segment structures by the developing lens.</a> Dev. Biol. 231, 63-76 (2001).</li><li>Neidhardt, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+screen+for+genes+controlling+mammalian+embryogenesis,+using+high-throughput+gene+expression+analysis+in+mouse+embryos.">Large-scale screen for genes controlling mammalian embryogenesis, using high-throughput gene expression analysis in mouse embryos.</a> Mech. Dev. 98, 77-94 (2000).</li><li>Gordon, J., Bennett, A. R., Blackburn, C. C., Manley, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gcm2+and+Foxn1+mark+early+parathyroid-+and+thymus-specific+domains+in+the+developing+third+pharyngeal.">Gcm2 and Foxn1 mark early parathyroid- and thymus-specific domains in the developing third pharyngeal.</a> 103, 141-143 (2001).</li></ol>

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 ...

Research

JoVE Journal

Biology

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Method for Whole Mount Antibody Staining in Chick

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...