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

Dit werk werd ondersteund door NIH subsidie ​​R21MH082360 (BGC) en R01HD056315 (NRM) en de Universiteit van Georgia.

1. Riboprobe generatie via in vitro transcriptie <ol><li> Voorbereiding PCR producten in vitro transcriptie templates. <ol><li> Designing PCR primers met faag transcriptie promoter sequenties in hun 5 uiteinden. Opmerking: De promotersequentie toegevoegd aan 5&#39;-uiteinde van de sense-streng PCR primer wordt gebruikt voor het transcriberen van de sense probe en de promotersequentie toegevoegd aan 5&#39;-uiteinde van de antisense PCR primer wordt gebruikt voor het synthetiser...

<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>

De methoden die in dit protocol zijn op basis van een aantal verschillende bronnen en geoptimaliseerd voor ganse berg E8.5-E11.5 dagen oude muis embryo&#39;s. Methoden voor het ganse berg in situ hybridisatie van gewervelde embryo&#39;s verscheen voor het eerst in de vroege jaren 1990 2,5-12. Dit protocol werd aangepast hoofdzakelijk van methoden ontwikkeld voor Xenopus embryo 7,8 en muis 2,11. In ons protocol veel nadruk wordt gelegd op zorgvuldige kwaliteitsbeoordeling van de ...

<table border="1"><tbody><tr><th> Naam </th><th> Type </th><th> Vennootschap </th><th> Catalogusnummer </th><th> Reacties </th></tr><tr><td> Digoxigenine-11-uridine-5&#39;-trifosfaat </td><td> Reagens </td><td> Roche </td><td> 11209256910 </td><td></td></tr><tr><td> Ribonucleoside trifosfaat Set </td><td> Reagens </td><td> Roche </td><td> 11277057001 </td><td></td></tr><tr><td> Snelle Draai Kolommen voor radioactief gelabelde RNA zuivering </td><td> Leveren </td><td> Roche </td><td> 11274015001 </td><td></td></tr><tr><td> T7 RNA polymerase </td><td> Reagens </td><td> Roche </td><td> 10881767001 </td><td></td></tr><tr><td> SP6 RNA polymerase </td><td> Reagens </td><td> Roche </td><td> 10810274001 </td><td></td></tr><tr><td> T3 RNA polymerase </td><td> Reagens </td><td> Roche </td><td> 11031163001 </td><td></td></tr><tr><td> CTP, [α-32P] - 800Ci/mmol 10mCi/ml, 250 uCi </td><td> Reagens </td><td> Perkin Elmer </td><td> BLU008X250UC </td><td></td></tr><tr><td> DNase I recombinant, RNase-free </td><td> Reagens </td><td> Roche </td><td> 4716728001 </td><td></td></tr><tr><td> Ureum, kleurloze tot witte kristallen of kristallijn poeder </td><td> Reagens </td><td> Visser </td><td> BP169-500 </td><td></td></tr><tr><td> Gel laadbuffer </td><td> Reagens </td><td> Ambion </td><td> AM8547 </td><td></td></tr><tr><td> Hybond-N +, Amersham </td><td> Leveren </td><td> GE Healthcare </td><td> RPN82B </td><td></td></tr><tr><td> UV Stratalinker 2400 </td><td> Uitrusting </td><td> Stratagene </td><td></td><td></td></tr><tr><td> Diethylpyrocarbonaat </td><td> Reagens </td><td> Sigma </td><td> D5758-100ML </td><td></td></tr><tr><td> Heparinenatrium </td><td&gt; Reagent </td><td> Acros </td><td> 41121-0010 </td><td></td></tr><tr><td> waterstofperoxide 30% in water </td><td> Reagens </td><td> Fisher Scientific </td><td> BP2633-500 </td><td></td></tr><tr><td> Gluteraldehyde, 8% EM graad </td><td> Reagens </td><td> Polysciences </td><td> 0/710 </td><td> Gebruik voorzichtig volgens vervaardiging instructies </td></tr><tr><td> Blokkerend reagens </td><td> Reagens </td><td> Roche </td><td> 11096176001 </td><td> Maak in 5% van de aandelen en bewaar bij -20 ° C </td></tr><tr><td> Proteinase K </td><td> Reagens </td><td> Roche </td><td> 3115852001 </td><td></td></tr><tr><td> RNase A </td><td> Reagens </td><td> Roche </td><td> 10109142001 </td><td> Maak als 10 mg / ml bouillon en bewaar bij -20 ° C. </td></tr><tr><td> RNase T1 </td><td> Reagens </td><td> Roche </td><td> 10109193001 </td><td></td></tr><tr><td> Ultrapure Formamide </td><td> Reagens </td><td> Invitrogen </td><td> 15515-026 </td><td></td></tr><tr><td> Levamisol hydrochloride </td><td> Reagens </td><td> ICN Biomedicals. Inc </td><td> 155228 </td><td></td></tr><tr><td> Ribonucleïnezuur van Torula gist, Type VI </td><td> Reagens </td><td> Sigma </td><td> R6625 </td><td> fenol / chloroform geëxtraheerd en geprecipiteerd meerdere malen, opnieuw gesuspendeerd in met DEPC dH2O en opgeslagen bij -20 ° C </td></tr><tr><td> Anti-digoxigenine-AP Fab fragmenten </td><td> Reagens </td><td> Roche </td><td> 11093274910 </td><td></td></tr><tr><td> BM Purple AP Substraat, precipiterende. Kant-en-klare oplossing. </td><td> Reagens </td><td> Roche </td><td> 11 442 074 001 </td><td></td></tr><tr><td> 4 ml, Clear, Gesloten Top, opslag Vial Convenience Kit </td><td> Leveren </td><td> Nationale wetenschappelijke </td><td> B7800-2 </td><td></td></tr><tr&gt; <td> Schud N Bake hybridisatie oven </td><td> Uitrusting </td><td> Boekel Wetenschappelijke </td><td> 136400 </td><td></td></tr><tr><td> Biopsie Sure-Tek </td><td> Leveren </td><td> Fisherbrand </td><td> 15-200-402C </td><td></td></tr><tr><td> Paraplast Plus weefsel inbedding medium </td><td> Reagens </td><td> Fisherbrand </td><td> 23-021-400 </td><td></td></tr><tr><td> Peel-A-Way wegwerp plastic weefsel inbedding mallen </td><td> Leveren </td><td> Polysciences </td><td> 18646A </td><td></td></tr><tr><td> Colorfrost Plus Objectglaasjes </td><td> Leveren </td><td> Fisherbrand </td><td> 12-550-17 </td><td></td></tr><tr><td> Nuclear Fast Red </td><td> Reagens </td><td> Sigma </td><td> N8002 </td><td></td></tr><tr><td> Cytoseal 60 </td><td> Reagens </td><td> Richard-Allan Wetenschappelijke </td><td> 8310-16 </td><td></td></tr></tbody></table>

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

Whole mount in situ hybridisatie is een zeer informatieve benadering voor het definiëren van genexpressie patronen in embryo&#39;s. De in situ hybridisatie procedures lang zijn en technisch veeleisend met meerdere belangrijke stappen die gezamenlijk bijdragen aan de kwaliteit van het eindresultaat. Dit protocol beschrijft in detail een aantal belangrijke kwaliteitscontrole stappen voor het optimaliseren van sonde etikettering en prestaties algemeen., Ons protocol geeft een gedetailleerde beschrijving van de kritische stappen die nodig zijn om reproduceerbaar te verkrijgen resultaten van hoge kwaliteit. Eerst beschrijven we de generatie van digoxigenine (DIG) gemerkte RNA probes via in vitro transcriptie van DNA templates gegenereerd door PCR. We beschrijven drie kritische kwaliteitscontrole assays om de hoeveelheid, integriteit en specifieke activiteit van het DIG gemerkte probes bepalen. Deze stappen zijn belangrijk voor het genereren van een probe voldoende gevoeligheid voor endogene mRNA te detecteren in een geheel muizenembryo.Daarnaast beschrijven we methoden voor de fixatie en opslag van E8.5-E11.5 dagen oude muizenembryo&#39;s in situ hybridisatie. Vervolgens beschrijven we Methoden voor beperkte proteinase K digestie van het gerehydrateerde embryo gevolgd door de gegevens van de hybridisatieomstandigheden, post-hybridisatie wassingen en RNase behandeling van niet-specifieke probe hybridisatie verwijderen. Een AP-geconjugeerd antilichaam wordt gebruikt om de gelabelde probe visualiseren en het expressiepatroon van de endogene transcript onthullen. Representatieve resultaten worden getoond van succesvolle experimenten en typische suboptimale experimenten.

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

Deze hele mount<em&gt; In situ</em&gt; Hybridisatie protocol bespreekt kritische stappen die reproduceerbare resultaten van hoge kwaliteit voor genexpressie studies te waarborgen in E8.5-E11.5 dagen oude muis embryo&#39;s.

Hele Mount<em&gt; In situ</em&gt; Hybridisatie van E8.5 aan E11.5 muis embryo&#39;s

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

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

Double Whole Mount in situ Hybridization of Early Chick Embryos

Dubbele Hele Mount in situ hybridisatie van het vroege embryo&#39;s Chick

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

Methode voor Whole Mount antilichaam kleuring in Chick

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Zebravis hele Mount High-Resolution Double Fluorescent In Situ Hybridisatie

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

Met behulp van Whole Mount In situ Hybridisatie aan Moleculaire Biologie en organismale Link

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

Een High Throughput In situ Hybridisatie Methode om mRNA expressie patronen karakteriseren in de Fetal Mouse lagere urogenitale tractus

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

Hele Mount RNA TL<em&gt; In situ</em&gt; Hybridisatie van<em&gt; Drosophila</em&gt; Embryo&#39;s

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

RNA<em&gt; In situ</em&gt; Hybridisatie in Hele Mount Embryo&#39;s en histologie Aangepast voor Marine kraakbeenvissen

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

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Visualisatie en analyse van mRNA moleculen met behulp van fluorescentie<em&gt; In Situ</em&gt; Hybridisatie in<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

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

Hoge resolutie Hele Mount<em&gt; In Situ</em&gt; Hybridisatie in zebravis embryo&#39;s om genexpressie en functie te bestuderen

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 ...