Name: in situ protocol for butterfly pupal wings using riboprobes
Uploaded: 2007-05-28T00:00:00
Description: Watch this Scientific Journal Video about In situ Protocol for Butterfly Pupal Wings Using Riboprobes at JoVE.com

Ringraziamo Jayne Selegue, Margaret Hollingsworth, Jin Berry, Ryo Futahashi, Najmus Sahar Mahfooz, Aleksandar Popadic, Bob Reed, Roche supporto tecnico per assistenza per la risoluzione di questo protocollo. Ringraziamo anche William Piel per consigli su come modificare il film.

GIORNO 1 <ol><li> Preparare le seguenti soluzioni: <ul><li> 10x PBS </li><li> 1x PBS </li><li> 1x PBT </li><li> DDH 2 O <ul><li> Aggiungi DEPC per lo 0,1% del volume totale e scuotere vigorosamente le soluzioni. </li><li> Autoclave. </li></ul></li><li> Fix paraformaldeide 4% - con PBS-DEPC </li><li> Soluzione di proteinasi K - presenza di un precipitato, vortice vigorosamente prima di rimuovere aliquota </li><li> Digestione di arresto Buffer </li><li> Prehybridization Buffer </li><li> 50:50 PBT: Buffer Preh...

<ol>
	<li>Carroll, S. B., Gates, J., Keys, D. N., Paddock, S. W., Panganiban, G. E. F., Selegue, J. E., Williams, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+formation+and+eyespot+determination+in+butterfly+wings.">Pattern formation and eyespot determination in butterfly wings.</a> Science. 265, 109-114 (1994).</li><li>Chomczynski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solubilization+in+formamide+protects+RNA+from+degradation.">Solubilization in formamide protects RNA from degradation.</a> Nucleic Acids Research. 20, 3791-3792 (1992).</li><li>Keys, D. N., Lewis, D. L., Selegue, J. E., Pearson, B. J., Goodrich, L. V., Johnson, R. J., Gates, J., Scott, M. P., Carroll, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruitment+of+a+hedgehog+regulatory+circuit+in+butterfly+eyespot+evolution.">Recruitment of a hedgehog regulatory circuit in butterfly eyespot evolution.</a> Science. 283, 532-534 (1999).</li><li>Marcus, J. M., Ramos, D. M., Monteiro, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ+line+transformation+of+the+butterfly+Bicyclus+anynana.">Germ line transformation of the butterfly Bicyclus anynana.</a> Proc R Soc Lond B (Suppl). 271, S263-S265 (2004).</li><li>Monteiro, A., Glaser, G., Stockslagger, S., Glansdorp, N., Ramos, 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=Comparative+insights+into+questions+of+lepidopteran+wing+pattern+homology.">Comparative insights into questions of lepidopteran wing pattern homology.</a> BMC Developmental Biology. 6, 52 (2006).</li><li>Ramos, D. M., Kamal, F., Wimmer, E. A., Cartwright, A. N., Monteiro, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+spatial+control+of+transgene+expresson+using+laser+induction+of+the+hsp70+promoter.">Temporal and spatial control of transgene expresson using laser induction of the hsp70 promoter.</a> BMC Developmental Biology. 6, 55 (2006).</li><li>Weatherbee, S. D., Nijhout, H. F., Grunert, L. W., Halder, G., Galant, R., Selegue, J., Carroll, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrabithorax+function+in+butterfly+wings+and+the+evolution+of+insect+wing+patterns.">Ultrabithorax function in butterfly wings and the evolution of insect wing patterns.</a> 9, 109-115 (1999).</li></ol>

Ottimizzazione dei protocolli esistenti In ibridazioni in situ su dischi ala larvali sono stati effettuati con successo con i dischi da Précis farfalle coenia (Carroll et al 1994;. Keys et al 1999;. Weatherbee et al 1999).. L&#39;attuale protocollo è stato adattato da un protocollo dettagliato scritto disponibili su richiesta presso il Laboratorio Carroll colorazioni ala larvale. I cambiamenti che abbiamo fatto servono ad accogliere le differenze tra i tessuti ala larvale e pupale. Durante la pupa dissezioni hindwing, la membrana ...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Fix Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>4% Paraformaldehyde in PBS</td>
</tr>
<tr>
<td>PBT</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>0.1% Tween 20 in PBS</td>
</tr>
<tr>
<td>Proteinase K solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>2.5&#xF06D;g/ml Proteinase K in PBT</td>
</tr>
<tr>
<td>Digestion Stop Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>2 mg/ml glycine in PBT</td>
</tr>
<tr>
<td>Pre-Hybridization Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For 50 ml PHB: 12 ml DEPC treated water + 25 ml Formamide + 12.5 ml 20 x SSC + 50 ul Tween 20 + 500 ul 10 mg/ml salmon sperm (Rnase free, heat denatured prior to addition to solution).
</td>
</tr>
<tr>
<td>Hybridization Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Add 1 mg/ml glycogen to prehybridization buffer</td>
</tr>
<tr>
<td>Block Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>50 mM Tris pH 6.8, 150 mM NaCl, 0.5% IGEPAL (NP40), 5 mg/ml BSA
</td>
</tr>
<tr>
<td>Anti-DIG</td>
<td>Ab</td>
<td>Roche Applied Science</td>
<td>11 093 274 910</td>
<td>Alkaline Phosphatase conjugated</td>
</tr>
<tr>
<td>DIG Wash and Block Buffer Set</td>
<td>&nbsp;</td>
<td>Roche Applied Science</td>
<td>11 585 762 001</td>
<td>&nbsp;</td>
<tr>
<td>Crystal Mount Aqueous Mounting Medium</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C0612</td>
<td>Mounting Medium </td>
</tr>		</tbody>
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in situ protocol for butterfly pupal wings using riboprobes

Qui vi presentiamo, in formato video, un protocollo per ibridazioni in situ in pupa ali della farfalla Bicyclus anynana utilizzando riboprobes. In ibridazioni in situ, un pilastro della biologia dello sviluppo, sono utili per studiare i modelli spaziali e temporali di espressione genica nello sviluppo di tessuti a livello della trascrizione. Se gli anticorpi che colpiscono i prodotti proteici di trascrizione del gene non sono ancora stati sviluppati, e / o ci sono copie del gene multiple di una particolare proteina del genoma che non possono essere differenziate utilizzando anticorpi disponibili, in situs può essere utilizzato in sostituzione. Mentre un a tecnica in situ per dischi ala larvale è stato a disposizione della comunità farfalla per diversi anni, l&#39;attuale protocollo è stato ottimizzato per le ali più grandi e più fragile pupa.

Watch this Scientific Journal Video about In situ Protocol for Butterfly Pupal Wings Using Riboprobes at JoVE.com

In situ Protocol for Butterfly Pupal Wings Using Riboprobes

Al fine di esaminare l&#39;espressione genica nel tessuto ala pupa di Bicyclus anynana, presentiamo un protocollo ottimizzato per ibridazioni in situ utilizzando riboprobes. Forniamo anche linee guida per l&#39;ottimizzazione di questo protocollo per l&#39;utilizzo in ali pupa di altre specie di lepidotteri.

Nel protocollo in situ per Ali di farfalla pupa Utilizzando Riboprobes

Here we present, in video format, a protocol for in situ hybridizations in pupal wings of the butterfly Bicyclus anynana using riboprobes.  In situ ...

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