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

Biz Jayne Selegue Margaret Hollingsworth, Jin Berry, Ryo Futahashi, Najmus Sahar Mahfooz, Aleksandar Popadiç Bob Reed, Roche Teknik Destek Bu protokol sorunları giderme konusunda yardım için teşekkür ederim. Biz de film düzenleme William Piel tavsiye için teşekkür ederim.

1. GÜN <ol><li> Aşağıdaki çözümleri hazırlayın: <ul><li> 10x PBS </li><li> 1x PBS </li><li> 1x PBT </li><li> GKD 2 O <ul><li> % 0.1 &#39;lik toplam hacmi DEPC ekleyin ve şiddetle çözümler sallamak. </li><li> Otoklav. </li></ul></li><li> % 4 Paraformaldehit Fix - PBS-DEPC </li><li> Proteinaz K çözüm çökelti varsa, vorteks şiddetle kısım çıkarmadan önce </li><li> Sindirim Durdur Tamponu </li><li> Prehybridization Tampon </li><li> 50:50 PBT: Prehybridization Tampon </li><li> Hibridizasy...

<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>

Mevcut protokollerin optimizasyonu Larva kanat disklerine yerinde döllemeler başarıyla Precis coenia kelebekler diskler kullanılarak yapılmıştır (Carroll ve ark 1994; Keys ve ark 1999; Weatherbee ve ark 1999). Protokolü larva kanat renklendirmeler Carroll Lab isteği üzerine ayrıntılı bir yazılı protokol adapte edilmiştir. Yapılan değişiklikler, larva ve pupa kanat dokular arasındaki farklılıkları barındırmak için hizmet vermektedir. Pupa hindwing diseksiyonlar sırasında peripodial membran kuyuları dahil...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<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>
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in situ protocol for butterfly pupal wings using riboprobes

Burada, video formatı, riboprobes kullanarak kelebek Bicyclus anynana pupa kanatları in situ döllemeler için bir protokol mevcut. Yerinde döllemeler, gelişim biyolojisi bir dayanak noktası, transkripsiyon düzeyinde, gelişmekte olan dokularda gen ekspresyonu mekansal ve zamansal desen çalışması için faydalıdır. Gen transkripsiyonu ve protein ürünleri hedef alan antikorlar henüz geliştirilen ve / veya mevcut antikorlar kullanılarak ayırt edilemez genomunda belli bir protein birden fazla gen kopyaları bulunmaktadır edilmemiştir varsa, situs yerine kullanılabilir. Larva kanat diskler için yerinde teknik bir kaç yıl için kelebek topluluk olmasına karşın, mevcut protokol daha büyük ve daha kırılgan bir pupa kanatlar için optimize edilmiştir.

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

Bicyclus anynana pupa kanat dokusu gen ekspresyonu incelemek için, riboprobes kullanarak yerinde döllemeler için optimize edilmiş bir protokol mevcut. Biz de bu protokolün diğer lepidopteran türlerin pupa kanatları kullanmak için daha fazla optimizasyon için yönergeler sağlar.

Riboprobes kullanarak Kelebek pupa Wings in situ Protokolü

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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