Name: double whole mount in situ hybridization of early chick embryos
Uploaded: 2008-10-27T00:00:00
Description: Watch this Scientific Journal Video about Double Whole Mount in situ Hybridization of Early Chick Embryos at JoVE.com

Dit werk werd ondersteund door de Margaret M. Alkek Stichting RHF.

Deel 1: Het bevestigen van de embryo&#39;s <ol><li> Vul dissectie schaal met ijskoud DEPC-PBS. Te houden op het ijs. Open het ei door te tikken op de schaal met een tang en verwijder de schil stukken. </li><li> Verwijder de dikke albumine met een pincet. Tilt dooierzak met grof tang zodat embryo naar boven wijst. </li><li> Met behulp van een schaar, knip een vierkant van dooierzak rond het embryo. </li><li> Met behulp van lepel, verwijder embryo van de dooier en de plaats op ijskoud DEPC-PBS. </li><li> ...

<ol>
	<li>Streit, A., Berliner, A. J., Papanayotou, C., Sirulnik, A., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+neural+induction+by+FGF+signaling+before+gastrulation.">Initiation of neural induction by FGF signaling before gastrulation.</a> Nature. 406, 74-78 (2000).</li><li>Rodr&#237;guez Esteban, C., Capdevila, J., Economides, A. N., Pascual, J., Ortiz, A., Izpis&#250;a Belmonte, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+novel+Cer-like+protein+Caronte+mediates+the+establishment+of+embryonic+left-right+asymmetry.">The novel Cer-like protein Caronte mediates the establishment of embryonic left-right asymmetry.</a> Nature. 401, 243-251 (1999).</li><li>Psychoyos, D., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+the+organizer+after+radical+ablation+of+Hensen's+node+and+the+anterior+primitive+streak+in+the+chick+embryo.">Restoration of the organizer after radical ablation of Hensen's node and the anterior primitive streak in the chick embryo.</a> Development. 122, 3263-3273 (1996).</li><li>Kawakami, M., Nakanishi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+an+endogenous+PKA+inhibitor,+PKIalpha,+in+organizing+left-right+axis+formation.">The role of an endogenous PKA inhibitor, PKIalpha, in organizing left-right axis formation.</a> Development. 128, 2509-2515 (2001).</li><li>Basch, M. L., Bronner-Fraser, M., Garcia-Castro, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+of+the+neural+crest+occurs+during+gastrulation+and+requires+Pax7.">Specification of the neural crest occurs during gastrulation and requires Pax7.</a> Nature. 441, 218-222 (2006).</li></ol>

De twee-kleuren ganse berg in situ hybridisatie methode wordt gebruikt om zowel de ruimtelijke en temporele patronen van genexpressie te bepalen, met behulp van een of twee genen van belang. Toepassingen zijn onder andere de beoordeling van genexpressie patronen van nieuwe genen (bijvoorbeeld 1,2, evenals veranderingen in genexpressie na belediging (embryonale manipulaties 3, 4 kralen, en elektroporatie van RNA-of DNA-constructen 5).

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Eggs</td>
<td>Animal</td>
<td>Charles River </td>
<td>Premium Fertile</td>
<td>&nbsp;</td>
<tr>
<td>Stereomicroscope</td>
<td>Microscope</td>
<td>Leica Microsystems</td>
<td>MZ9.5</td>
<td>&nbsp;</td>
<tr>
<td>Marsh Automatic Incubator</td>
<td>Tool</td>
<td>Lyon</td>
<td>RX</td>
<td>&nbsp;</td>
<tr>
<td>Hybridization Incubator</td>
<td>Tool</td>
<td>Hybaid</td>
<td>Micro-4</td>
<td>&nbsp;</td>
<tr>
<td>Adams Nutator orbital mixer</td>
<td>Tool</td>
<td>Fisher</td>
<td>14-062</td>
<td>&nbsp;</td>
<tr>
<td>Curved Forceps (1)</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>72991-4C</td>
<td>&nbsp;</td>
<tr>
<td>Fine scissors</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>14161-10</td>
<td>&nbsp;</td>
<tr>
<td>Spoon</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>10370-18</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur Capillary Pipette</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>70950-12</td>
<td>&nbsp;</td>
<tr>
<td>Microcapillary tube</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1049-1PAK</td>
<td>Pull using vertical micropipette puller; blunt end with fine forceps</td>
</tr>
<tr>
<td>Microdissecting knife</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>10056-12</td>
<td>Use to puncture cavities prior to in situ hybridization</td>
</tr>
<tr>
<td>Minuten pins 0.2mm diam</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26002-20</td>
<td>Keep pins together using a magnetic stirrer;</td>
</tr>
<tr>
<td>Sylgard 184 Silicon Elastomer Curing Agent and Base</td>
<td>Reagent</td>
<td>Dow Corning</td>
<td>0001986475</td>
<td>Mix 1 part Curing Agent, 9 parts Base; set O/N at 37C</td>
</tr>
<tr>
<td>Diethylpyrocarbonate (depc)</td>
<td>Reagent</td>
<td>Acros Organics</td>
<td>10025025</td>
<td>Add 1ml depc to 1l PBS; shake; autoclave</td>
</tr>
<tr>
<td>16% PFA</td>
<td>Reagent</td>
<td>Electron Microscopy Sciences</td>
<td>15710</td>
<td>&nbsp;</td>
<tr>
<td>Tween-20</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P1379-100ML</td>
<td>&nbsp;</td>
<tr>
<td>Proteinase K</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P2308-5MG</td>
<td>Stock at 10 mg/ml in depc-H2O in 10ul aliquots. Store at -20C.</td>
</tr>
<tr>
<td>Formamide, deionized</td>
<td>Reagent</td>
<td>Ambion</td>
<td>9342</td>
<td>&nbsp;</td>
<tr>
<td>Yeast tRNA-25 mg</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15401-011</td>
<td>Stock at 20 mg/ml in depc-H2O in 125ul aliquots. Store at -20C</td>
</tr>
<tr>
<td>Heparin Sodium salt</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H4784-250MG</td>
<td>Stock at 50 mg/ml in depc-H2O in 100ul aliquots. Store at -20C.</td>
</tr>
<tr>
<td>SSC x20 pH5.5</td>
<td>Reagent</td>
<td>Fisher</td>
<td>PR-V4261</td>
<td>&nbsp;</td>
<tr>
<td>EDTA (0.5M)</td>
<td>PR-V4231</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>SDS</td>
<td>Reagent</td>
<td>Fisher</td>
<td>BP166-100</td>
<td>&nbsp;</td>
<tr>
<td>Maleic acid</td>
<td>Reagent</td>
<td>Fluka</td>
<td>63189</td>
<td>&nbsp;</td>
<tr>
<td>Boehringer Blocking Reagent</td>
<td>Reagent</td>
<td>Roche Applied Sciences</td>
<td>11096176001</td>
<td>Use at 2% in MABT. To make, heat with MABT for 20 mn at 60C. Can make a stock and store in 1-5 ml aliquots at 20C.</td>
</tr>
<tr>
<td>Heat inactivated sheep serum</td>
<td>Reagent</td>
<td>Jackson Immuno</td>
<td>013-000-121</td>
<td>Dissolve in 10 ml H2O; heat 550C, 30 mn. Aliquot at 1ml, -20C.</td>
</tr>
<tr>
<td>Anti-Digoxigenin-AP, Fab fragments</td>
<td>Reagent</td>
<td>Roche Applied Sciences</td>
<td>11093274910</td>
<td>&nbsp;</td>
<tr>
<td>NBT/BCIP stock solution</td>
<td>Reagent</td>
<td>Roche Applied Sciences</td>
<td>11681451001</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Fluorescein-AP, Fab fragments</td>
<td>Reagent</td>
<td>Roche Applied Sciences</td>
<td>11426338910</td>
<td>&nbsp;</td>
<tr>
<td>2M Tris</td>
<td>BP1759-500</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vector Red Alkaline Phosphatase Substrate Kit</td>
<td>Reagent</td>
<td>Vector Laboratories</td>
<td>SK-5100</td>
<td>&nbsp;</td>
<tr>
<td>1,2,3,4-tetrahydronaphthalene</td>
<td>Reagent</td>
<td>Sigma</td>
<td>429325-100ML</td>
<td>Under hood</td>
</tr>		
</tbody>
</table>
</div>

double whole mount in situ hybridization of early chick embryos

Het kuiken embryo is een waardevol instrument in de studie van de vroege embryonale ontwikkeling. De transparantie, toegankelijkheid en het gemak van manipulatie, maken het een ideaal hulpmiddel voor het bestuderen van genexpressie in de hersenen, neurale buis, somiet en hart primordia vorming. Deze video toont de verschillende stappen in 2 kleuren ganse berg in situ hybridisatie, Ten eerste is de embryo wordt ontleed door het ei en gefixeerd in paraformaldehyde. Ten tweede, is het embryo verwerkt voor prehybridisatie. Het embryo wordt vervolgens gehybridiseerd met twee verschillende probes, gekoppeld aan een DIG, en een gekoppeld aan FITC. Na &#39;s nachts hybridisatie, is het embryo geïncubeerd met DIG gekoppeld antilichaam. Kleur reactie voor DIG substraat wordt uitgevoerd, en de regio van belang lijkt blauw. Het embryo wordt vervolgens geïncubeerd met FITC gekoppeld antilichaam. Het embryo wordt verwerkt voor de kleurreactie met FITC, en de regio van belang lijkt rood. Tot slot wordt het embryo vast en verwerkt voor phtograph en snijden. Een gids is het oplossen van problemen ook gepresenteerd.

Watch this Scientific Journal Video about Double Whole Mount in situ Hybridization of Early Chick Embryos at JoVE.com

Double Whole Mount in situ Hybridization of Early Chick Embryos

Deze video toont twee-kleuren ganse berg in situ hybridisatie, een methode waarmee de ruimtelijke en temporele expressie patroon van twee verschillende genen kunnen worden gevisualiseerd in jonge kippenembryo&#39;s. Deze methode werd oorspronkelijk geïntroduceerd door David Wilkinson, Domingos Henrique, Phil Ingham en David Ish-Horowicz.

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