Patrick Cafferty Kanada Multipl Skleroz Derneği bir doktora sonrası burs tarafından desteklenmektedir.

Bölüm 1: Ön-deneysel set-up. <ol><li> Bir hafta önceden arkadaşı larva oluşturmak için uçar ifade GFP kontrolü altında özel bir glial organizatörü. Deneme için bir nükleer yerelleştirme sırası ile ters polarite (repo) organizatörü (3, 4) glial hücreleri kullanarak ifade etiketli GFP görüntülendi. </li><li> % 1 gecede kuru poli-L-lisin çözüm ve hava 10 dakika boyunca 18 mm yuvarlak kapak fişleri iliklerine tarafından en az bir gün önceden fişleri kapak hazırla. </li><li> Deney bir gün önce, bir gecede% 70 etanol bileşenleri iliklerine Chamlide manyetik kültür odası temiz. </li><li> Deney günü, Schneider böcek orta aseptik teknik kullanılarak 10 ml fetal sığır serumu, penisilin-streptomisin çözüm, ve insülin ekleyerek kültür ortamı hazırlamak. Çalışma konsantrasyonları: 1X fetal sığır serum; 100U/ml penisilin, 0.1mg/ml streptomisin ve 0.2 mg / ml Schneider böcek ortamda insülin (değiştirilmiş (5)). </li><li> Kültür odasının bir hücre kültürü kaputu kurumaya bırakın. Hazırlanan kültür ortamı ile durulama kültür odası artık etanol yıkayınız. </li></ol> Bölüm 2: Drosophila göz-beyin kompleks Diseksiyon. <ol><li> Birkaç dakika süreyle buz üzerinde soğutulmuş kültür ortamı bir petri açılan bir sinek şişe ve yerin yan tarafında bir üçüncü instar dolaşıp larva seçin. Larva Chilling diseksiyon kolaylığı için peristaltik vücut kasılmaları yavaş tavsiye edilir. </li><li> Diseksiyon mikroskop altında Sylgard kaplı çanak kültür ortamı bir damla soğuk larva yerleştirin. </li><li> Diseksiyon mikroskobu altında, larva arka ucundan yolu yaklaşık üçte birini sıkıca kavramak için Dumont ince forseps bir çift kullanın. Larva ön sonundan itibaren ağzında kanca extrudes kadar bekleyin. Ağız tam uzantısı üzerine ikinci bir çift forseps ile kanca kavrayın. Larva incelemek için, forseps, iki çift zıt yönlerde yavaşça çekin. Göz-beyin kompleksi, tükürük bezleri, yağ organları, ve hayali doku ile birlikte, larva vücuttan uzakta çeker. Ağız, göz-beyin kompleks yırttınız hızla aksi takdirde çok kanca çekmeyin. </li><li> Tükürük bezleri, yağ organları, ve ultra-ince clipper makas kullanılarak hayali diskler uzak kesin. Beyin ve göz-diskler iki yarımküresinin ventral sinir kablosu ve ağız çengelleri eklenecektir. </li><li> 18 mm kapak kayma bir cam slayt üzerine yerleştirin. Sonra kapak kayma pick up için slayt kenarına asılı kapak kayma bir kenar bırakın. 18 mm kapak kayma kültür ortamı üzerine bir damla ekleyin. Forseps ile ağız kanca tutarak, kültür ortamı içine göz-beyin kompleksi aktarın. Forseps veya doku hasar ile doğrudan göz-beyin kompleksi kavramak etmeyin. </li><li> Ince diseksiyon makas kullanmak, ağız, göz-beyin kompleksi ve atın kanca keserek. Ağız kancaları, doku mikroskopi sırasında taşımak için neden kültür ortamı sözleşme devam edecek gibi ağız kanca kaldırılması canlı görüntüleme için önemlidir. </li><li> Göz hayali disk içinde glial göç görselleştirmek için, dikkatlice kesilmiş ve optik sapı, beyin ve göz disk bağlayan ince bir doku (Şekil 1A) beyin uzağa itmek ya da atın. Optik sapı içindeki glia görselleştirmek için, beyin, optik sapı ve sağlam göz hayali disk (Şekil 1E) bırakın. </li><li> Forseps kullanarak lamel Pick up ve kalıcı bir kalem ile ilgi doku etrafında altında bir daire çizin. Bu daire Bölüm 4 mikroskobu için doku yerini yardımcı olacaktır. </li></ol> Bölüm 3: Manyetik bir kültür odasında göz hayali disk montajı. <ol><li> Forseps kullanarak, lamel asma kenarında kavrayın. Doku kültürü bozmadan Chamlide manyetik odasının alt plaka lamel aktarın. </li><li> Chamlide odasının ana gövde üzerine silikon O-ring yerleştirin. Alt plakası ana gövde üstüne yükleyin. </li><li> Odaya kültür ortamı yavaş yavaş ekleyin. Kültür ortamında çok hızlı ya da ilgi doku rahatsız olacak katmayın. Kapağın altında gaz değişimi için izin odası tamamen doldurmayın. </li><li> Chamlide odasının üstünde görünmez kapağını yavaşça yerleştirin. </li></ol> Bölüm 4: göz disk glial hücreleri göç Görselleştirme. <ol><li> Konfokal veya floresan mikroskobu aşamasında kültür odasına yerleştirin. Düşük büyütme bir referans olarak lamel daire kullanarak örnek bulun. </li><li> 40x bir lens kullanarak örnek üzerinde odaklanın. Doku lamel üzerine yerleşmek için izin verin. Göz disk veya optik sapı 3-4 saat boyunca her 10-15dk görüntüler yakalayın. Doku seğirme ve odak dışında hareket edebilir. El görüntü yakalama önce doku odaklanarak inci hafifletmekbir sorundur. </li></ol> Bölüm 5: Temsilcilik sonuçları: Doğru şekilde gerçekleştirilmemesi, protokol bize göz hayali disk (Şekil 1 BD) optik sapı göç GFP etiketli glial hücreleri görüntüler bir dizi toplamak için izin verdi. 60 dakikalık bir süre için canlı görüntüleme, vahşi bir tür göz hayali disk içinde glial çekirdeğinin pozisyonlarda değişiklikleri gözlemlemek için yeterli iken, glial hücre geçiş için gerekli bir genin mutant glial çekirdekleri tamamen optik sapı (oklar çıkmak için başarısız Şekil 1 FH). Biz kültür dokusunun bozulmasına gözlemleyerek önce 240 dakika gibi uzun süreler için kültürlü göz-beyin kompleksleri var. 240 dakikalık zaman noktası, GFP pozitif hücrelerin kültürlü göz-beyin kompleksler (ok Şekil 2 C) çevreleyen kültür ortamında gözlemlemek için başlar. Ayrıca GFP boyunca diffüz noktalar bir arıza doku bütünlüğü düşündüren dokularda birikir. <img src="/files/ftp_upload/1155/figure1.png" alt="" width="589" height="241" /> Şekil 1: gelişmekte olan yabani tip ve mutant ve görüntü sistemleri GFP etiketli glial çekirdekleri Canlı görüntüleme. A, E) Görüntüler diferansiyel girişim kontrast (DIC), kültürlü yabani tip ve mutant göz hayali diskler (ED) mikroskopi kullanılarak çekilen . Vahşi tip, glial hücreler doğumlu ve göz-disk, optik sapı (OS) göç. Beyin, düzleşme ve göz hayali disk görüntüleme kolaylaştırmak için kaldırılmıştır. BD) (A) 0, 30 ve 60 dakikalık zaman noktalarında GFP etiketli glial çekirdekleri göz disk içinde göç ortaya koymaktadır vahşi tip kültür göz hayali disk Floresan mikroskopi. FH) (E) 0, 30 ve 60 dakikalık zaman içinde GFP etiketli glial çekirdekleri durdurduklarını optik sapı (ok) gösteren mutant göz-beyin kompleks Floresan mikroskopi. (BR) beyin sapı içinde glia görüntüleme kolaylaştırmak için optik sapı bağlı bırakılmıştır. <img src="/files/ftp_upload/1155/Figure_2.png" alt="" width="600" height="156" /> Şekil 2: Canlı görüntüleme vahşi bir tür göz hayali disk GFP etiketli glial membranlarda. Biz başarıyla 240 dakikalık süreler için göz-beyin kültür kompleksleri var. Doku kültürü 240 dakikadan daha uzun yıkmak için başlar. 300 dakikalık bir zaman noktası (C) ile karşılaştırıldığında göz hayali disk çevresindeki kültür ortamı (ED), optik sapı (OS) ve 0 (A) ve 150 dakika (B) beyin lobu (BR), ücretsiz GFP pozitif hücreler, bir ok ile gösterilir.

<ol>
	<li>Aigouy, B., Lepelletier, L., Giangrande, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+chain+migration+requires+pioneer+cells.">Glial chain migration requires pioneer cells.</a> J. Neurosci. 28, 11635-11641 (2008).</li><li>Silies, M., Yuva, Y., Engelen, D., Aho, A., Stork, T., Klambt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cell+migration+in+the+eye+disc.">Glial cell migration in the eye disc.</a> J. Neurosci. 27, 13130-13139 (2007).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Sepp, K. J., Auld, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+lacZ+enhancer+trap+lines+to+GAL4+lines+using+targeted+transposition+in+Drosophila+melanogaster.">Conversion of lacZ enhancer trap lines to GAL4 lines using targeted transposition in Drosophila melanogaster.</a> Genetics. 151, 1093-1101 (1999).</li><li>Gibson, M. C., Patel, A. B., Nagpal, R., Perrimon, 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+emergence+of+geometric+order+in+proliferating+metazoan+epitheia.">The emergence of geometric order in proliferating metazoan epitheia.</a> Nature. 442, 1038-1041 (2006).</li><li>Lee, T., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+analysis+with+a+repressible+cell+marker+(MARCM)+for+Drosophila+neural+development.">Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development.</a> Trends Neurosci. 24, 251-254 (2001).</li></ol>

The authors have nothing to disclose.

Bu protokol canlı mikroskopi kullanılarak göz hayali disk glial hücre göç gözlem açıklar. Vahşi tip örnek (Şekil 1 MS), bir saat boyunca göz disk glial hücre hareketi gözlemlemek için bir nükleer GFP işareti kullanılır. Şu anda bizim laboratuvarda çalışma kapsamında, glial hücre göçü için gerekli bir aday gen için bir mutant, biz (Şekil 1 EH) bir saatlik bir süre boyunca bir optik sapı içinde glial hücre çekirdeğinin durdurduklarını gözlemlenmiştir. Bizim stratejimiz il...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Poly-L-lysine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P8920</td>
<td>&nbsp;</td>
<tr>
<td>Schneider's Insect Media</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S0146</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P4458</td>
<td>&nbsp;</td>
<tr>
<td>Insulin solution from bovine pancreas</td>
<td>Reagent</td>
<td>Sigma</td>
<td>I0516</td>
<td>&nbsp;</td>
<tr>
<td>Chamlide Magnetic chamber</td>
<td>Tool</td>
<td>Live cell Instrument</td>
<td>CM-R-10</td>
<td>35 mm dish type chamber for 18 mm coverslip</td>
</tr>
<tr>
<td>Ultra fine clipper scissors</td>
<td>Tool</td>
<td>Fine scientific tools</td>
<td>15200-00</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5 forceps</td>
<td>Tool</td>
<td>Fine scientific tools</td>
<td>11251-20</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent microscope</td>
<td>Microscope</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>Any fluorescent imaging system that has the necessary filters and excitation for GFP can be used. </td>
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live imaging of glial cell migration in the drosophila eye imaginal disc

Omurgalı ve omurgasız organizmaların hem de glial hücreleri ensheath için nihai hedef bölgelere göç ve ilişkili nöronlar desteği gerekir. Son ilerleme gelişmekte olan pupa kanat (1), çalışmaları glial hücreler canlı göç tanımlamak için yapılmış olsa da<em&gt; Drosophila</em&gt; Glial hücre göçü genellikle sabit dokusunun incelenmesi yer var. Canlı hareketli hücrelerin mikroskobik inceleme, oranı ve büyüme yönünde değişiklikler belirlenmesi de dahil olmak üzere göçmen süreci boyunca hücresel davranışlarını incelemek için yeteneği sunar. Genetik araçlarının kullanımı ile eşleştirilmiş, canlı görüntüleme, kalkınma sürecinde belirli genler için daha hassas rollerini belirlemek için kullanılabilir. Silies tarafından Önceki çalışma<em&gt; Ve ark.</em&gt; (2), sabit dokusunda göz hayali disk, optik sapı, gelişmekte olan beyin ve göz bağlayan bir yapı kaynaklanan göç glia tanımladı. Burada, glial hücrelerin canlı göç incelenmesi için bir protokol taslağını<em&gt; Drosophila</em&gt; Göz hayali disk. Biz gelişmekte olan glia yabani tip ve mutant hayvanlar glial hücre ilerlemesini takip etmek, GFP ifade eden Drosophila hattı yararlanmak.

Watch this Scientific Journal Video about Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc at JoVE.com

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Burada, glial hücreler göç GFP etiketli glial hücreler ile eşleştirilmiş canlı mikroskobik analizi ile gelişmekte olan Drosophila göz içine incelemek için bir protokol açıklar.

Drosophila Göz hayali Disk Glial Hücre Göç Canlı Görüntüleme

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

live-imaging-glial-cell-migration-drosophila-eye-imaginal

Research

JoVE Journal

Biology

10.6K Görüntüleme.  University of British Columbia - UBC. Burada, glial hücreler göç GFP etiketli glial hücreler ile eşleştirilmiş canlı mikroskobik analizi ile gelişmekte olan Drosophila göz içine incelemek için bir protokol açıklar.

Video: Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

3. Instar Drosophila Larva hayali Disklerin Diseksiyon

Biyoloji

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

Canlı görselleştirme Drosophila</emFloresan Boyalar> Glial nöromüsküler Kavşağı

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

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 ...

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries made using this system have taken advantage of the spatiotemporal nature of photoreceptor differentiation in the developing eye imaginal disc. To use this system it is first necessary for the researcher to learn to identify and dissect the eye disc. We describe a novel RFP reporter to aid in the identification of the eye disc and the visualization of specific cell types in the developing eye. We detail a methodology for dissection of the eye imaginal disc from third instar larvae and describe how the eye-RFP reporter can aid in this dissection. This eye-RFP reporter is only expressed in the eye and can be visualized using fluorescence microscopy either in live tissue or after fixation without the need for signal amplification. We also show how this reporter can be used to identify specific cells types within the eye disc. This protocol and the use of the eye-RFP reporter will aid researchers using the Drosophila eye to address fundamentally important biological questions.

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

We describe a novel red fluorescent protein (RFP) reporter that is expressed specifically in the Drosophila eye. We detail a methodology for dissection of the eye imaginal disc and how this reporter can be used to aid in the dissection and identification of specific cell types in the developing eye.

Göz hayali Disk Görselleştirme Yardım Roman RFP Reporter Drosophila</em

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

Uygulamalarda Canlı Görüntüleme Drosophila melanogaster Embriyonik Hemocyte Göçler

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

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 ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

GFP etiketli Proteinlerin Görüntüleme Canlı<em&gt; Drosophila</em&gt; Oosit

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Apoptotik hücresi Temizliği görüntülenmesi sırasında Canlı<em&gt; Drosophila</em&gt; Embriyo

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

Mozaik Yetişkin Canlı Görüntüleme Domates / GFP-FLP / FRT Yöntemi<em&gt; Drosophila</em&gt; Fotoreseptör Hücreler

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Dissection and Mounting of Drosophila Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

Diseksiyon ve montajı<em&gt; Drosophila</em&gt; Pupa Göz Diskler

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...