Susan Gerbi Heidi Smith ve David Lambert, sırasıyla, Sciara coprophila ve Ilyanassa obsoleta in vivo santrifüj test etmek için malzeme temini için teşekkür ederiz . Biz Welte laboratuar tekniği ve el yazması üzerinde yorum yapmak için üye teşekkür ederiz. In vivo santrifüj testin geliştirilmesi ve arıtma MAW NIGMS hibe GM64687 tarafından desteklenmiştir. Görüntüleri Şekil. 1 ve Şekil. 5E, F, daha önce 2 basıldı ve izni ile yeniden basıldı.

<ol>
	<li>Wieschaus, E., N&#252;sslein-Volhard, C., Roberts, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+at+embryos.">Looking at embryos.</a> Drosophila: A Practical Approach. , 179-214 (1998).</li><li>Cermelli, S., Guo, Y., Gross, S. P., Welte, 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=The+lipid+droplet+proteome+reveals+that+droplets+are+a+protein+storage+depot.">The lipid droplet proteome reveals that droplets are a protein storage depot.</a> Curr Biol. 16, 1783-1795 (2006).</li><li>Kiehart, D. P., Crawford, J. M., Montague, R. A., Sullivan, W., Ashburner, M., Hawley, 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=Quantitative+microinjection+of+Drosophila+embryos.">Quantitative microinjection of Drosophila embryos.</a> Drosophila Protocols. , 345-359 (2000).</li><li>LaJeunesse, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+new+Drosophila+markers+of+intracellular+membranes.">Three new Drosophila markers of intracellular membranes.</a> Biotechniques. 36 (784-788), 790-790 (2004).</li><li>Schuh, M., Lehner, C. F., Heidmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+Drosophila+CID/CENP-A+and+CENP-C+into+centromeres+during+early+embryonic+anaphase.">Incorporation of Drosophila CID/CENP-A and CENP-C into centromeres during early embryonic anaphase.</a> Curr Biol. 17, 237-243 (2007).</li><li>Clarkson, M., Saint, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+His2AvDGFP+fusion+gene+complements+a+lethal+His2AvD+mutant+allele+and+provides+an+in+vivo+marker+for+Drosophila+chromosome+behavior.">A His2AvDGFP fusion gene complements a lethal His2AvD mutant allele and provides an in vivo marker for Drosophila chromosome behavior.</a> DNA Cell Biol. 18, 457-462 (1999).</li><li>Welte, M. A., Gross, S. P., Postner, M., Block, S. M., Wieschaus, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+regulation+of+vesicle+transport+in+Drosophila+embryos:+forces+and+kinetics.">Developmental regulation of vesicle transport in Drosophila embryos: forces and kinetics.</a> Cell. 92, 547-557 (1998).</li><li>Tran, S. L., Welte, 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=unpublished+observations.">unpublished observations.</a> , .</li><li>Rothwell, W. F., Sullivan, W., Sullivan, W., Ashburner, M., Hawley, 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=Fluorescent+analysis+of+Drosophila+embryos.">Fluorescent analysis of Drosophila embryos.</a> Drosophila Protocols. , 141-157 (2000).</li><li>Guo, Y., Jangi, S., Welte, 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=Organelle-specific+Control+of+Intracellular+Transport:+Distinctly+Targeted+Isoforms+of+the+Regulator+Klar.">Organelle-specific Control of Intracellular Transport: Distinctly Targeted Isoforms of the Regulator Klar.</a> Mol Biol Cell. 16, 1406-1416 (2005).</li><li>Meyer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overlapping+Functions+of+Argonaute+Proteins+in+Patterning+and+Morphogenesis+of+Drosophila+Embryos.">Overlapping Functions of Argonaute Proteins in Patterning and Morphogenesis of Drosophila Embryos.</a> PLoS Genet. 2, 1224-1239 (2006).</li><li>Shubeita, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+motor+copy+number+on+the+intracellular+transport+of+kinesin-1-driven+lipid+droplets.">Consequences of motor copy number on the intracellular transport of kinesin-1-driven lipid droplets.</a> Cell. 135, 1098-1107 (2008).</li><li>Welte, 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=Regulation+of+lipid-droplet+transport+by+the+Perilipin+homologue+LSD2.">Regulation of lipid-droplet transport by the Perilipin homologue LSD2.</a> Curr. Biol. 15, 1266-1275 (2005).</li><li>Phadnis, N., Welte, 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=Unpublished+Observations.">Unpublished Observations.</a> , .</li><li>Phadnis, N., Welte, 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=Unpublished+Observations.">Unpublished Observations.</a> , .</li><li>Morgan, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+XXII:+The+redistribution+of+the+visible+materials+of+the+egg+by+centrifuging.">Chapter XXII: The redistribution of the visible materials of the egg by centrifuging.</a> Experimental Embryology. , (1927).</li><li>Welte, 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=Unpublished+Observations.">Unpublished Observations.</a> , .</li><li>Bownes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+oogenesis+and+embryogenesis+resulting+from+centrifuging+Drosophila+melanogaster+females.">Abnormal oogenesis and embryogenesis resulting from centrifuging Drosophila melanogaster females.</a> J Embryol Exp Morphol. 40, 65-81 (1977).</li></ol>

Kritik adımlar Agar agar konsantrasyonu yeterince yüksek olduğundan emin olun. Çok düşükse, santrifüj sırasında parçalanır ve embriyoların patlaması ya da rastgele odaklı olacak . Agar konsantrasyonu sadece biraz agar (4.3 adım aşırı AKM yeterince kurutulmuş veya yetersiz kaldırma) ıslak çok düşük ya da eğer varsa, santrifüj sırasında embriyoların deliklerinden dışarı float ve yanlış odaklı hale gelecektir. Agar çok ince dökülür, aynı zamanda çökmesi veya santrifüj sırasında çatlak. Organelleri, tek tek hücrelerin binlerce içinde tecrit olacak embriyolar cellularization aşamalarında ötesinde gelişmiş olduğunu emin olun, aksi takdirde, organellerin ayrılması (Şekil 5C) iş olmaz . Bir hücre aşamada genç embriyoların, organeller büyük mesafeler göç ücretsiz ve onların karakteristik yoğunluğuna göre birikir. Çok eski olan embriyolar analiz önlemek için, emin olun, (i) kadın, eski yumurta (adım 2.4), (ii) (adım 2.5) 3 saat veya daha az embriyolar toplamak, (iii) bırakmak için izin vermek için bir ön toplama agar içine gömmek için görsel cellularization aşamalarında önce embriyolar seçin (adım 4.2), (iv) cellularization (adım 4.6) sonuna kadar gelişimini önlemek için kısa bir süre gömme tutmak, ve (v) (adım 5.6) doğru katmanı yoktu embriyolar iptal . Santrifüj zaman eksik etmeyin. Bazı embriyoların sadece 10 dakika santrifüj sonra bile güzel katmanlarını göstermek rağmen, ayırma embriyo embriyoya tutarsızdır. 30 dakika spin yüksek tutarlı sonuçlar verir. Ayrıca, santrifüj sona erdikten sonra uzun süre santrifüj süresi daha uzun katmanları stabildir. Bu kullanmadan önce daha fazla embriyoların süreci zaman alır, bu uygulamalar için önemlidir (örneğin, nakli deneyleri için konfokal analizi için işlenen veya monte edilmesi gerekiyorsa). Olası değişiklikler Yumurta toplama ve adım 2 ve 3 1, 3, 9 koryon kaldırılması çok farklı protokoller var ve kullanılan embriyoların genç olanlar sürece burada açıklanan yanı sıra iş, yumurta verimi yüksek ve kesir yanlış sahnelenen embriyolar en aza indirilir. Ön-arka hizalama protokol posterior uç agar gömüldü ve ön ucu (Şekil 3) olduğu gibi tüm embriyolar yönlendirir. Bu yönelim, tutarlılık için iyi micropyle santrifüj (Şekil 1, 5) sırasında sivri ucunu bir dönüm noktası olarak kullanmak mümkündür, böylece. Ancak, uygulama ile, parlak alan mikroskobu ile lipit damlacık ve yumurta sarısı katmanları farklı bir görünüm santrifüje embriyoların yönünü ortaya çıkarmak kolaydır. Sonra iki ucunda olan embriyolar monte etmek mümkün olur; bu esneklik montaj sürecini hızlandırır. Yönlenimsiz santrifüj protokolün en çok zaman alıcı ve teknik açıdan zor yönü montaj agar embriyolar (adım 4). Bu iki kez (bir kez deliğe takın ve bir kez santrifüj sonra kurtarmak için) her bir embriyo dokunmadan gerektirir. Alternatif olarak, bir AKM ile dolu mikrosantrifüj tüpler içine adım 4.1 &#39;embriyolar transfer edebilirsiniz. Bu borular, mikrosantrifüj (13.000 rpm, 10-30 dak.) Santrifüj, embriyoları aynı zamanda tipik tabakası 10-13 . Bu değişim, bir çok hızlı bir şekilde aynı anda yüzlerce embriyo süreci sağlar. Ancak, katman olarak tutarlı değildir; embriyoların bir kısmını yukarıda protokol uygulanan daha yüksek santrifüj kuvvetler (~ 16.000 g) rağmen farklı katmanlar ortaya çıkmaz. Daha zorlu embriyoların yönünü değişkendir ve bir büyük embriyo ekseni çeşitli açılardan ayrımı izleyebilirsiniz gerçektir. Bu değişkenlik, deneyci, belirli bir embriyo santrifüj tüpüne nasıl düzenlenmiştir santrifüj sonra dışarı sıralamak için gereklidir. Sarısı otofloresans embriyonun en yoğun bölümü için bir belirteç olarak kullanımı ile güvenilir bir şekilde bunu yapmak için çok daha pratik ve daha fazla karar gerektirir rağmen, bu, çoğu zaman mümkündür. Örnek yeterli embriyolar varsa, bir embriyolar Şekil gibi düzenlenmelidir oldu bu örnekleri sıralamak mümkün olabilir. 1. Kendi yumurta kabuğu embriyoların Santrifüj koryon ilk 2 çıkarmadan adım 4 agar embriyolar gömmek için mümkündür. Bu yaklaşımda, toplama plakası üzerinde embriyolar Halokarbon yağ yerine adım 3.2 (Halokarbon koryon saydam döner)% 50 çamaşır suyu ile kaplıdır. Embriyolar uygun aşamalarında seçilmiş ve cımbız kullanarak yeni bir agar plaka transfer; cımbız, zarar, sadece yumurta filamentler kapmaembriyonun düzgün kaçınılmalıdır. Embriyolar ön yumurta filamentler agar delikten dışarı yapıştırma ile, 4.8 ile adım 4.4 gibi gömülürler. Embriyolar santrifüj ve adım 5.4 agar çıkardı sonra yumurta kabuğu,% 50 çamaşır suyu arıtma ile kaldırıldı. Yumurta kabuğu varlığında Santrifüj fiksasyon sonra devitellinization oranı (adım 6.2) artırabilir, ama santrifüj için doğru aşamalarında seçimi (4.2 adım) daha zordur. Uygulamalar Embriyo santrifüj ko deneyler, yani, eğer belli bir proteinin belli bir büyük organel lokalize sınamak için yararlıdır. Örneğin, Klar protein erken embriyonik lipid damlacıkları bulunan, henüz bu lokalizasyon sağlam embriyolar göstermek zordur. Bölümünde, Klar puncta ve lipid damlacıkları hem de sabit sahte ko ekarte etmek için, embriyo çevre 10 bol çünkü bu. Ancak, farklı bir tabaka haline santrifüj lipid damlacıkları konsantreleri, ve böylece, Klar sinyal ile ko 10 belirginleşmektedir. Benzer analiz, belirli histon gibi şaşırtıcı örnekler de dahil olmak üzere lipid damlacıkları 8, 13-15, 2 ve bir protein yayg mevcut olduğu durumlarda diğer proteinlerin lokalizasyonu net bir şekilde gösterdi, ancak lipid damlacıkları 8 zenginleştirilmiş. Embriyolarının gelişim aşamasında (adım 4.2) tarafından görsel olarak seçilebilir olarak, lipid damlacıkları 8, 15 proteinlerin gelişimsel düzenlenir işe tespit etmek bile mümkün . Çünkü görsel olarak ayrı bir &quot;kap&quot; lipid damlacıkları birikir lipid damlacıkları ile ko için test özellikle kolaydır, böylece başka marker veya leke bu katmanı tanımlamak için gereklidir. Aslında, çünkü kolaylığı ve potansiyel çarpıcı görsel sonuçları (Şekil 1, Şekil 5 D, E), bizim laboratuar aday protein, lipid damlacıkları lokalize olup olmadığını bir ilk test olarak artık rutin olarak bu tekniği kullanır. Prensip olarak, benzer ko çalışmaları Şekil diğer organellerin için çalışması gerekir. 1, muhtemelen dağıtım santrifüje embriyolar diğerleri (hücre içi parazitler de dahil olmak üzere) belgelenmelidir henüz için. Santrifüj sıkı bantlar organelleri konsantreleri bu yana, bu organeller zenginleştirilmiş bir kısmını izolasyonu kolaylaştırır. Bu yaklaşım, zaten içine donör embriyoların 2 nakli için lipid damlacıkları kurtarmak için kullanılır olmuştur. Ayrıca zenginleştirilmiş fraksiyonu (örneğin, 15 fiksasyon sonra embriyolar kapalı katmanları keserek) biyokimyasal analiz etmek mümkün olabilir. Santrifüj 16 (kurbağa, nematod, gömlekliler, halkalı, deniz kestaneleri, yumuşakçalar ve cnidarians da dahil olmak üzere) pek çok farklı türlerin yumurta tabakalandırmak için kullanılabilir yöntemi, burada Drosophila embriyolar ötesinde uygulamalar vardır anlatılan. Kendi laboratuarında, housefly Musca domestica 2 embriyolar için yukarıdaki protokol ve mantar sivrisineği fosili Sciara coprophila 17 ve salyangoz Ilyanassa obsoleta 17 embriyoların analiz yönlenimsiz santrifüj istihdam. Son olarak, bu yöntem embriyolar sınırlı değildir: Santrifüj oosit (örneğin, Drosophila 2, 18 ve sütlü özsuyu olan bir bitki hata Oncopeltus fasciatus 17) gibi diğer büyük hücreler, sınıflandırmaktı yararlı olabilir. Farklı örnek için, tam santrifüj koşulları optimize edilmesi gerekir: Örneğin, 3000 g 10 dakika verimli Musca domestica 2 embriyolar tabakalandırmak için yeterli ve eğer çok daha uzun santrifüj, embriyoların fiksasyon ve immün sırasında dağılma eğilimi gösterir.

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		<div>
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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>60x15 mm Petri dishes</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>#35-1007</td>
<td>From Falcon</td>
</tr>
<tr>
<td>Wire mesh</td>
<td>&nbsp;</td>
<td>Small Parts</td>
<td>CX-0150</td>
<td>stainless steel type 304; mesh size #150 x150, wire diameter: 0.0026 inches</td>
</tr>
<tr>
<td>Tungsten needles</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>10130-10</td>
<td>0.25 mm</td>
</tr>
<tr>
<td>Moria Nickel Plated Pin/Needle Holder</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26016-12</td>
<td>&nbsp;</td>
<tr>
<td>Fly Cages</td>
<td>&nbsp;</td>
<td>Genesee Scientific</td>
<td>59-100 or 59-101</td>
<td>For 65 mm or 100 mm diameter Petri dishes, respectively</td>
</tr>
<tr>
<td>Halocarbon Oil 27</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H8773</td>
<td>&nbsp;</td>		</tbody>
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in-vivo centrifugation of drosophila embryos

Batmaz yoğunluğu farklılıkları onları birbirinden ayırmak için farklı tipte hücre içi organelleri arındırıcı ve izole etmek için önemli bir strateji. Ancak, hücreler santrifüj, proteinler ve yerli olmayan bu ortamda organelleri önce kesintiye çoğu zaman uygunsuz birbirine yapışmaz. Burada, ayrı organelleri sağlam yaşayan Drosophila embriyolar yoğunluk bir metodu tanımlar . Cellularization önce Erken embriyoların nüfus kafeslerden hasat edilir ve dış yumurta kabukları,% 50 çamaşır suyu ile tedavi kaldırılır. Embriyolar sonra agar küçük dikey deliklere, ilk önce küçük bir agar plaka ve eklenen arka sonuna aktarılır. Gömülü embriyoları içeren plakalar 3000g az 30 dakika süreyle santrifüj. Agar embriyoların destekler ve belirli bir yönde tutmaktadır. Daha sonra, embriyolar agar künt bir iğne ile çıkardı. Santrifüj bir tabakalaşma parlak alan mikroskobu ile kolayca görülebilir, farklı katmanlar halinde büyük organelleri ayırır. Floresan belirteçlerin bir dizi canlı embriyolarının başarılı tabakalaşma onaylamak için kullanılabilir. Proteinler belli organelleri ile ilgili ko gösteren belirli bir tabaka güçleniyor. Bireysel katmanlar, donör yumurta içine biyokimyasal analiz veya transplantasyon için telafi edilebilir. Bu teknik, farklı türlerin yumurta ve oosit dahil olmak üzere diğer büyük hücre, organel ayrımı için geçerlidir.

Watch this Scientific Journal Video about In-vivo Centrifugation of Drosophila Embryos at JoVE.com

In-vivo Centrifugation of Drosophila Embryos

Biz yaşayan organelleri yoğunluğu ile ayrı bir metodu tanımlar<em&gt; Drosophila</em&gt; Embriyolar gibi. Embriyolar agar gömülü ve santrifüj. Bu teknik, ön-arka embriyo eksen boyunca büyük organellerin tekrarlanabilir ayrılığı ortaya çıkarır. Bu yöntem, biyokimyasal analiz ve nakli deneyleri için ko deney ve verim organel kesirler kolaylaştırır.

In-vivo Drosophila Embriyolar Santrifüj

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

in-vivo-centrifugation-of-drosophila-embryos

Research

JoVE Journal

Biology

In-vivo Centrifugation of Drosophila Embryos

18.3K Görüntüleme.  University of Rochester. Biz yaşayan organelleri yoğunluğu ile ayrı bir metodu tanımlar Drosophila Embriyolar gibi. Embriyolar agar gömülü ve santrifüj. Bu teknik, ön-arka embriyo eksen boyunca büyük organellerin tekrarlanabilir ayrılığı ortaya çıkarır. Bu yöntem, biyokimyasal analiz ve nakli deneyleri için ko deney ve verim organel kesirler kolaylaştırır.

Video: In-vivo Centrifugation of Drosophila Embryos

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Nöronal Kültürler Midgastrula Sahne Drosophila Embriyolar hazırlanması

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Biyoloji

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

Hazırlanması Drosophila</emAsma Bırak Protokolü kullanarak Canlı Görüntüleme> Embriyolar

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

Mezoskopik Floresans Tomografi için In-vivo Görüntüleme Geliştirme Drosophila</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Elektron Mikroskobu için embriyo Hazırlık Drosophila Embriyonik kalp tüpü

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

VE Dik Görüntüleme Drosophila Embriyolar

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Birincil Hücre Kültürleri Drosophila Gastrula Embriyolar

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

Alt hücresel Çözünürlük bozulmamış Drosophila Larva in vivo Görüntüleme

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

DsRNA Enjeksiyon RNAi Girişim içine Drosophila Embriyolar

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

Living Görüntüleme Hücre Şekli Değiştir Drosophila Embriyolar

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Kinesin İzolasyonu ve Saflaştırılması gelen Drosophila Embriyolar

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...