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

Detta arbete stöddes av NIH bidrag R21MH082360 (BGC) och R01HD056315 (NRM) samt University of Georgia.

1. Riboprob generation genom in vitro-transkription <ol><li> Förberedelse PCR-produkter för in vitro transkription mallar. <ol><li> Designing PCR-primrar med promotor fag transkriptions-sekvenser i deras 5-ändar. Obs: Promotorsekvensen läggs till 5&#39;-änden av sens-strängen PCR-primer kommer att användas för att transkribera känslan sonden, och promotorsekvensen sattes till 5&#39;-änden av antisens-PCR-primern kommer att användas för att syntetisera anti-sens sö...

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De metoder som beskrivs i detta protokoll har anpassats från ett antal olika källor och optimerad för hela montering E8.5-E11.5 dygn gamla musembryon. Metoder för hela montera in situ hybridisering av ryggradsdjur embryon visades först i början av 1990-talet 2,5-12. Detta protokoll anpassades främst från metoder som utvecklats för Xenopus embryon 7,8 samt mus 2,11. I vår protokoll stor vikt läggs på noggrann kvalitetsbedömning av sonden. Noggrann uppmärksamhet att ...

<table border="1"><tbody><tr><th> Namn </th><th> Typ </th><th> Företag </th><th> Katalognummer </th><th> Kommentarer </th></tr><tr><td> Digoxigenin-11-uridin-5&#39;-trifosfat </td><td> Reagens </td><td> Roche </td><td> 11209256910 </td><td></td></tr><tr><td> Ribonukleosid trifosfat Set </td><td> Reagens </td><td> Roche </td><td> 11277057001 </td><td></td></tr><tr><td> Quick Spin Kolumner för radiomärkt RNA-rening </td><td> Supply </td><td> Roche </td><td> 11274015001 </td><td></td></tr><tr><td> T7 RNA-polymeras </td><td> Reagens </td><td> Roche </td><td> 10881767001 </td><td></td></tr><tr><td> SP6 RNA-polymeras </td><td> Reagens </td><td> Roche </td><td> 10810274001 </td><td></td></tr><tr><td> T3 RNA-polymeras </td><td> Reagens </td><td> Roche </td><td> 11031163001 </td><td></td></tr><tr><td> CTP, [α-32P] - 800Ci/mmol 10mCi/ml, 250 pCi </td><td> Reagens </td><td> Perkin Elmer </td><td> BLU008X250UC </td><td></td></tr><tr><td> DNas I rekombinant, RNas-fri </td><td> Reagens </td><td> Roche </td><td> 4716728001 </td><td></td></tr><tr><td> Urea, färglös till vita kristaller eller kristallint pulver </td><td> Reagens </td><td> Fisher </td><td> BP169-500 </td><td></td></tr><tr><td> Gelladdningsbuffert </td><td> Reagens </td><td> Ambion </td><td> AM8547 </td><td></td></tr><tr><td> Hybond-N +, Amersham </td><td> Supply </td><td> GE Healthcare </td><td> RPN82B </td><td></td></tr><tr><td> UV Stratalinker 2400 </td><td> Utrustning </td><td> Stratagene </td><td></td><td></td></tr><tr><td> Dietylpyrokarbonat </td><td> Reagens </td><td> Sigma </td><td> D5758-100ML </td><td></td></tr><tr><td> Heparinnatrium </td><td&gt; Reagens </td><td> Acros </td><td> 41121-0010 </td><td></td></tr><tr><td> väteperoxid 30% i vatten </td><td> Reagens </td><td> Fisher Scientific </td><td> BP2633-500 </td><td></td></tr><tr><td> Gluteraldehyd, 8% EM grade </td><td> Reagens </td><td> Polysciences </td><td> 0/710 </td><td> Använd med försiktighet enligt tillverkarens anvisningar </td></tr><tr><td> Blockeringsreagens </td><td> Reagens </td><td> Roche </td><td> 11096176001 </td><td> Göra till 5% lager och butik vid -20 ° C </td></tr><tr><td> Proteinas 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> Gör som 10 mg / ml lager och butik vid -20 ° C. </td></tr><tr><td> RNas T1 </td><td> Reagens </td><td> Roche </td><td> 10109193001 </td><td></td></tr><tr><td> Ultrapure Formamid </td><td> Reagens </td><td> Invitrogen </td><td> 15515-026 </td><td></td></tr><tr><td> Levamisol hydroklorid </td><td> Reagens </td><td> ICN Biomedicals. Inc </td><td> 155.228 </td><td></td></tr><tr><td> Ribonukleinsyra från Torula jäst, typ VI </td><td> Reagens </td><td> Sigma </td><td> R6625 </td><td> fenol / kloroform extraherades flera gånger och utfälldes, resuspenderades i DEPC-dH2O och lagrades vid -20 ° C </td></tr><tr><td> Anti-digoxigenin-AP Fab-fragment </td><td> Reagens </td><td> Roche </td><td> 11093274910 </td><td></td></tr><tr><td> BM Lila AP substrat, utfällning. Klar att använda lösningen. </td><td> Reagens </td><td> Roche </td><td> 11 442 074 001 </td><td></td></tr><tr><td> 4 ml, Klar, sluten topp, lagring Injektionsflaska Convenience Kit </td><td> Supply </td><td> Nationella vetenskapliga </td><td> B7800-2 </td><td></td></tr><tr&gt; <td> Shake n Bake hybridisering ugn </td><td> Utrustning </td><td> Boekel vetenskapliga </td><td> 136.400 </td><td></td></tr><tr><td> Biopsi Sure-Tek </td><td> Supply </td><td> Fisherbrand </td><td> 15-200-402C </td><td></td></tr><tr><td> Paraplast Plus vävnad inbäddningsmedium </td><td> Reagens </td><td> Fisherbrand </td><td> 23-021-400 </td><td></td></tr><tr><td> Peel-A-Way disponibel plast vävnad inbäddning formar </td><td> Supply </td><td> Polysciences </td><td> 18646A </td><td></td></tr><tr><td> Colorfrost Plus objektglas </td><td> Supply </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 Scientific </td><td> 8310-16 </td><td></td></tr></tbody></table>

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

Hela montera in situ hybridisering är en mycket informativ strategi för att definiera mönster genuttryck i embryon. In situ hybridisering förfaranden är långa och tekniskt krävande med flera viktiga steg som tillsammans bidrar till kvaliteten på det slutliga resultatet. Detta protokoll beskriver i detalj flera kontroll viktiga kvalitets steg för att optimera sond märkning och prestanda. Sammantaget ger vår protokoll en detaljerad beskrivning av de kritiska åtgärder som är nödvändiga för att reproducerbart få resultat av hög kvalitet. Först beskriver vi generering av digoxigenin-märkta (DIG) RNA-prober via transkription in vitro av DNA-mallar genererades genom PCR. Vi beskriver tre kritiska analyser kvalitetskontroll för att bestämma mängden, integritet och specifik aktivitet för DIG-märkta prober. Dessa steg är viktiga för att generera en sond med tillräcklig känslighet för att detektera endogena mRNA i en hel musembryo.Dessutom beskriver vi metoder för fixering och lagring av E8.5-E11.5 dygn gamla musembryon för in situ hybridisering. Sedan beskriver vi detaljerade metoder för begränsad proteinas K nedbrytning av de rehydrerade embryon följt av detaljerna i de hybridiseringsbetingelser, efter hybridisering tvättar och RNas behandling för att avlägsna icke-specifik sond hybridisering. En AP-konjugerad antikropp används för att visualisera den märkta proben och avslöjar uttrycksmönstret av den endogena transkriptet. Representativa resultat visas från framgångsrika experiment och typiska suboptimala experiment.

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

Hela denna montering<em&gt; In situ</em&gt; Hybridisering protokoll diskuterar viktiga steg som säkerställer reproducerbara resultat av hög kvalitet för studier genuttryck i E8.5-E11.5 dygn gamla musembryon.

Hela berget<em&gt; In situ</em&gt; Hybridisering av E8.5 till E11.5 musembryon

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

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

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