Yazarlar SI Bernstein (San Diego Eyalet Üniversitesi) Bu yazının hazırlanması ile ilgili eleştirel okuma ve yararlı önerileri için teşekkür ederim. Bu çalışma SI Bernstein, SDSU, R. Bodmer, BIMR NIH hibe programı tarafından desteklenen ve G. Batılı Devletler, Amerikan Kalp Derneği Ortaklık Vogler ve A. Cammarato bir post-doktora bursu.

Başlamadan önce <ol><li> Aşağıdaki çözümleri hazırlayın: <ol class="lalpha"><li> rahatlatıcı tamponu (10 mM EGTA içeren yapay Drosophila hemolimf (ADH) (&quot;Drosophilia&#39;daki kalpte görselleştirme&quot; bölümüne bakın)) </li><li> fiksatif (1x PBS içinde% 4 formaldehit) </li><li> PBSTx (% 0.1 Triton X-100 içeren PBS) </li><li> PBSTx uygun şekilde seyreltilmiş birincil ve türe spesifik floresan etiketli sekonder antikorlar </li></ol></li><li> Drosophila teşrih kalp tüpleri (erişkin sinek ve / veya larva) maruz <ol class="lalpha"><li> yarı sağlam Drosophila kalp hazırlanması &quot;Drosophila kalpte görselleştirme&quot; protokolü, veya </li><li> Drosophila Larvaların NMJ Diseksiyon aşağıdaki değişiklikler protokolü 1: Kullanım larva diseksiyonu sırasında ADH oksijenli ve ventral orta hat posterior iğne hafifçe yerinden. Larva ventral orta hat boyunca kesilir. Fiksasyon için önce herhangi bir doku çıkarmayın. </li></ol></li></ol> Floresan Boyama <ol><li> Tüm kalpleri ritmik oksijenli ADH yenerek olduğunu kontrol edin. Hızlı tampon rahatlatıcı ADH değiştirin. Kasılmaları inhibe edilir sağlamak için her kalp tüp inceleyin. </li><li> Fiksatif ile rahatlatıcı bir tampon yerine kalbini düzelt. Nazik sallayarak 20 dakika oda sıcaklığında inkübe edin. (Larva hazırlık çalkalanması için herhangi bir adım gerekli değildir ve kardiyak doku bütünlüğü için zararlı olarak kabul edilebilir). </li><li> On dakika boyunca sürekli titriyor oda sıcaklığında PBSTx örnekleri üç kez yıkayın. </li><li> Yetişkinler için, kalan şey daha oval ve daha az yuvarlak olduğu gibi karın manikür ventral kenarlar dikkatle geri düzeltin. Ayrıca, karın ve göğüs arasındaki tek kesim, dikkatli ve temiz ayrı vücut bölümlerinin ve kalbin ön bölgeye en az zararla sağlar. Larva kalpleri için, dikkatle, ince forseps kullanarak yağ vücut çıkarın. Larva kalp, özellikle kırılgan ve diğer kasların veya bağ dokusu çok az destek beri yağ alınması, son derece dikkatli yürütülmelidir. Bu kalp zarar verebilir olarak trakeal dallar çıkarmayın. </li><li> 50-100 ul PBSTx içinde seyreltilmesi birincil antikor içeren kuyu ile 96 kuyu plakasına, merkezi bir konumda yer alan kalp tüpü ile temastan kaçınarak, kenarlarından kesilmiş sırt bölgesi karın manikür aktarın. Başına en fazla 12 numuneler yerleştirin. Sürekli ajitasyon ile 2 saat oda sıcaklığında inkübe edin. Kuluçka de gecede 4 yapılabilir ° C. </li><li> Primer antikor çözümü çıkarın. 100 oda sıcaklığında sürekli sallayarak ul PBSTx on dakika boyunca kalpleri üç kez yıkayın. </li><li> Son yıkama tamponu kaldırılması ardından, Alexa594-Phalloidin (1:1000) ile desteklenmiş PBSTx 100 ul, ikincil antikor ekleyin. Oda sıcaklığında sürekli sallayarak bir saat süreyle inkübe edin. Örnekleri beyazlatma fluorofor önlemek için kapalı tutun. </li><li> Ikincil inkübasyon sonrasında oda sıcaklığında sürekli sallayarak PBSTx 100 ul ile on dakika boyunca kalpleri üç kez yıkayın. Örnekleri yıkama adımları boyunca kapalı tutun. </li><li> Triton X-100 kaldırılması için, 10 dakika süreyle PBS 100 ul içindeki kalpler kör olur son bir kez yıkayın. Örnekleri monte etmeden önce birkaç gün süreyle 4 ° C&#39;de karanlıkta saklanabilir. </li></ol> Yetişkin Kalpler Montaj <ol><li> 10 ul Vectashield montaj orta bir mikroskop lamı iki 18 x 18 mm kapak fişleri uyun. Lamelleri ~ 10-15 mm arayla olmalıdır. Iki lamelleri arasında orta montaj küçük bir üçüncü damla yerleştirin. </li><li> Üçüncü bir lamel merkezinde Vectashield 20 ul yerleştirin. </li><li> Manikür aşırı kenarlarından dikkatlice PBS yıkama solüsyonu her kalplerin kaldırmak ve yavaşça üçüncü lamel üzerine montaj orta damla kalp tarafı aşağı yerde. Lamel başına en fazla beş damla kalpleri yerleştirin. </li><li> Bütün kalpleri aşağı bakacak şekilde sağlamak için bir mikroskop altında kontrol edin. </li><li> Kalpleri içeren lamel dikkatlice ters ve hızlı lamelleri çifti içeren, kalp tüpleri sigortalar ile lamel çifti arasında Vectashield damlacık damlacık. Slaytta yer &quot;Köprü&quot; lamelleri tarafından oluşturulmalıdır. Kalpleri bir lamel ve mikroskop lamı arasında askıya alınacaktır. </li><li> Kalpleri şimdi yukarı gelecek sağlamak için bir mikroskop altında kontrol edin. </li><li> Lamelleri oje ile kenarlarına sabitleyin. </li><li> Kalpleri şimdi floresan veya konfokal mikroskobu aracılığıyla görüntülü olarak hazırız. </li></ol><ol><li> Bir mikroskop lamı üzerine bir damla (~ 20 ul) Vectashield yerleştirin. </li><li> Tungsten iğneler kullanarak aşağı Vectashield ve yönlendirmek onları dorsal tarafına içine iki larva örneklerinin dikkatle kadar veri transferi. </li><li> Bireysel montaj orta örneklerin dışına sürükleyin ve paralel olarak her hizalamak. </li><li> Örneğinin karşı taraf bir lamel yerleştirin. Forseps kullanarak, arka lamel bir tarafta döşeme ve böylece bir köprü oluşturan ön lamel aşağı düşürerek ilk kez, üst üste üçüncü bir lamel yerleştirin. Kılcal güçleri larva kalbin düzgün hizalama yardımcı anterior posterior Vectashield akışı neden olacaktır. </li><li> Oje, kenarlarında yer Fix tüm lamelleri ve dikkatli bir şekilde 20-30 ul Vectashield lamelleri arasındaki boşluğu doldurmak. </li><li> Mühür oje ve mağaza ile 4 ° C veya -20 ° C&#39;de uzun süreli depolama için </li></ol> Temsilcisi Sonuçlar Doğru çalıştırıldığında, dorsal geminin tüm bileşenleri ve ilgili dokuların sağlam kalır ve kolayca görüntülenebilmekte. Eşiğe düzeyde olmalıdır. Çok iyi yetişkin, ventral longitudinal kas tabakası lekeleri için önemli bir sinyal üretir (Şekil 1 ve Şekil 2). Altta yatan dairesel kardiyomiyositlerde Bununla birlikte, ventral örten tabakanın olduğu gibi yoğun bir sinyal üretmek için değil eğilimindedir. Yetişkin kalp ön konik odasına &quot;miyositler kontraktil malzemenin önemli bir miktarda içerir ve dolayısıyla, bu bölgede kalp tüp kalan en sağlam göreceli olarak görünür. Larva kalp yetişkin kontraktil kardiyomiyositlerde (Şekil 3) benzer bir helezonik myofibrillar düzenleme gösterir. <img src="/files/ftp_upload/1423/1423fig1atmb.jpg" alt="Şekil 1a" border="0" /> <img src="/files/ftp_upload/1423/1423fig1btmb.jpg" border="0" alt="Şekil 1b" /> Şekil 1: Üst. Gösteren bir yetişkin bir Drosophila melanogaster ifade miyozin-GFP tüm kalp tüp floresan mikrografı. Görüntü Apotome bir kayma modülü ile donatılmış bir Zeiss Imager Z1, floresan mikroskop ile alınmıştır. Miyozin-GFP yeşil gösterilir, aktin AlexaFluor ® 594 Phalloidin (kırmızı) ve α-aktinin ile boyandı anti-α-aktinin antikor (mavi) ile etiketlenir. Hazırlık prosedürü iyi korunmuş yapıları ile kalp örneklerinin kurtarma izin unutmayın. CC = konik kamara; AM = alary kas, V = dahili valf; VLL = ventral uzunlamasına kas tabakası. Alt. 3. sadece kasılma kardiyomiyositlerde helezonik myofibril düzenleme gösteren ventral longitudinal tabakasının altındaki kalp karın segment üzerinden CC yetişkin kardiyak tüpün bir bölge. Lütfen <a href="https://www.jove.com/files/ftp_upload/1423/1423fig1a.jpg">buraya tıklayın</a> Şekil 1a daha büyük bir versiyonu için, <a href="https://www.jove.com/files/ftp_upload/1423/1423fig1b.jpg">burada</a> daha büyük bir rakam 1b sürümü için <img border="0" src="/files/ftp_upload/1423/1423fig2tmb.jpg" alt="Şekil 2" /> Şekil 2: AlexaFluor ® 594 Phalloidin (kırmızı) ve bir anti-Pericardin (kollajen tip IV) (yeşil) antikor ile boyandı yetişkin kalp anterior kısmı Temsilcisi konfokal yığınları. Pericardin muhtemelen ventral kas tabakası uzunlamasına odaklı myofibrils kaynaklanan, ventral yüzeyi boyunca kalp ile ilişkilidir. Lütfen <a href="https://www.jove.com/files/ftp_upload/1423/1423fig2.jpg">buraya tıklayın</a> Şekil 2 daha büyük bir versiyonu için. <img border="0" src="/files/ftp_upload/1423/1423fig3tmb.jpg" alt="Şekil 3" /> Şekil 3: Bir floresan segment bir sahne üç Drosophila larva uygun kalp A7 mikrografı. Görüntü Apotome bir kayma modülü ile donatılmış bir Zeiss Imager Z1, floresan mikroskop ile alınmıştır. Aktin AlexaFluor ® 594 Phalloidin (yeşil) ve α-aktinin ile etiketlenmiş anti-α-aktinin antikor (kırmızı) ile boyandı. Lütfen <a href="https://www.jove.com/files/ftp_upload/1423/1423fig3.jpg">buraya tıklayın</a> daha büyük bir şekil 3 sürümü için.

<ol>
	<li>Brent, J. R., Werner, K. M., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+larval+NMJ+dissection.">Drosophila larval NMJ dissection.</a> J Vis Exp. , (2009).</li><li>Molina, M. R., Cripps, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ostia,+the+inflow+tracts+of+the+Drosophila+heart,+develop+from+a+genetically+distinct+subset+of+cardial+cells.">Ostia, the inflow tracts of the Drosophila heart, develop from a genetically distinct subset of cardial cells.</a> Mech Dev. 1, 51-59 (2001).</li><li>Dulcis, D., Levine, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamatergic+innervation+of+the+heart+initiates+retrograde+contractions+in+adult+Drosophila+melanogaster.">Glutamatergic innervation of the heart initiates retrograde contractions in adult Drosophila melanogaster.</a> J Neurosci. 2, 271-280 (2005).</li><li>Zeitouni, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signalling+pathways+involved+in+adult+heart+formation+revealed+by+gene+expression+profiling+in+Drosophila.">Signalling pathways involved in adult heart formation revealed by gene expression profiling in Drosophila.</a> PLoS Genet. 10, 1907-1921 (2007).</li><li>Robertson, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metamorphosis+of+Drosophila+melanogaster,+including+an+accurately+timed+account+of+the+principal+morphological+change.">The metamorphosis of Drosophila melanogaster, including an accurately timed account of the principal morphological change.</a> J. Morphol. 2, 351-399 (1936).</li><li>Miller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The internal anatomy and histology of the imago of Drosophila melanogaster. , (1950).</li><li>Rizki, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+circulatory+system+and+associated+cells+and+tissues.">The circulatory system and associated cells and tissues.</a> , (1978).</li><li>Cammarato, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+transducer+mutations+differentially+affect+motor+function,+myofibril+structure,+and+the+performance+of+skeletal+and+cardiac+muscles.">Myosin transducer mutations differentially affect motor function, myofibril structure, and the performance of skeletal and cardiac muscles.</a> Mol Biol Cell. 2, 553-562 (2008).</li><li>Taghli-Lamallem, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dystrophin+deficiency+in+Drosophila+reduces+lifespan+and+causes+a+dilated+cardiomyopathy+phenotype.">Dystrophin deficiency in Drosophila reduces lifespan and causes a dilated cardiomyopathy phenotype.</a> Aging Cell. 2, 237-249 (2008).</li><li>Kelso, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+database+documenting+a+GFP+protein-trap+insertion+screen+in+Drosophila+melanogaster.">a database documenting a GFP protein-trap insertion screen in Drosophila melanogaster.</a> Nucleic Acids Res. 32, D418-D420 (2004).</li><li>Dietzl, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+transgenic+RNAi+library+for+conditional+gene+inactivation+in+Drosophila.">A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.</a> Nature. 7150, 151-156 (2007).</li><li>Mery, 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+Drosophila+muscle+LIM+protein,+Mlp84B,+is+essential+for+cardiac+function.">The Drosophila muscle LIM protein, Mlp84B, is essential for cardiac function.</a> J Exp Biol. 211, 15-23 (2008).</li></ol>

Burada Drosophila melanogaster dorsal damar ve görüntüleme için floresan veya konfokal mikroskopi ile ilgili dokuların hazırlanması ve boyanması için yararlı bir protokol mevcut. Rafine edilmiş ve yaygın olarak larva ve yetişkin Drosophila kalp tüpleri in situ görüntüleme iyi bir çözüme izin verir etkin boyama laboratuarımızda istihdam adımların kısa ve öz bir açıklama sağlamalıdır . Diğerleri kısaltılmış biçim, 2, 3, 4 benzer yöntemler var. </p...

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<td>Ethylene glycol-bis (2-amino-ethylether) -N,N,N&#x2019;,N&#x2019;-tetra-acetic acid (EGTA)</td>
<td>Reagent</td>
<td>Sigma-Aldrich&reg;</td>
<td>E4378</td>
<td>&nbsp;</td>
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<td>Formaldehyde, 10%, methanol free, Ultra Pure</td>
<td>Reagent</td>
<td>Polysciences Inc.</td>
<td>50-00-0</td>
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<td>Triton-X-100</td>
<td>Reagent</td>
<td>Sigma-Aldrich&reg;</td>
<td>9002-93-1</td>
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<td>Alexa Fluor&reg; 594 phalloidin</td>
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<td>Vectashield&reg; Mounting Medium for Fluorescence with DAPI</td>
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<td>Vector Laboratories, Inc.</td>
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fluorescent labeling of drosophila heart structures

<em&gt; Drosophila melanogaster</em&gt; Dorsal damar, kalp, kasılma kardiyomiyositlerde, yetişkinlerde kalp duvarı, destekleyici alary kasları ve, ventral uzunlamasına kas hücrelerinin bir katman her tarafında hizalamak perikardiyal hücreleri tek bir tabaka oluşan tübüler bir yapıdır. Kasılma lifleri ev myofibrils ve homolog ekstraselüler matriks bileşenleri ile etkileşim sitoskeletal / submembranous protein kompleksleri, yoğun paketlenmiş demetleri de dahil olmak üzere kas hücre mimarisini bileşenlerinin korunmuş. Burada disseke larva ve yarı sağlam yetişkin kalbini fiksasyon ve özellikle miyokard elemanlarının floresan etiketleme için bir protokol tarif<em&gt; Drosophila</em&gt;. Özellikle larva kalplerinde sarkomerik F-aktin ve α-aktinin etiketleme göstermektedir. Ayrıca, vahşi tip yetişkin kalplerinde, yetişkin sinekler ifade ve α-aktinin ve pericardin, tip IV ekstraselüler matriks kollajen F-aktin ve α-aktinin miyozin-GFP etiketlenmesi. Özellikle dikkat taşıma en aza indirir ve kalp tüpleri ve ilgili dokuların bütünlüğünü korumak için fırsat optimize yarı sağlam yetişkin kalpleri için bir montaj stratejisi verilir. Bu hazırlıklar, floresan ve konfokal mikroskobu ile görüntüleme için uygundur. Genel olarak, bu yordamı dikkatli ve ayrıntılı bir analiz için güçlü bir genetik çözülebilir bir model sistem kalbin yapısal özellikleri sağlar.

Watch this Scientific Journal Video about Fluorescent Labeling of Drosophila Heart Structures at JoVE.com

Fluorescent Labeling of Drosophila Heart Structures

Burada larva ve yetişkin kalp tüplerin farklı unsurları floresan etiketlenmesi için temel bir protokol tanımlamak<em&gt; Drosophila melanogaster</em&gt;. Bu örnekler, floresan veya konfokal mikroskopi ile görüntüleme için çok uygundur. Bu teknik, güçlü bir model organizma kalplerin özellikleri ayrıntılı yapısal analiz izin verir.

VE Floresan Etiketleme Drosophila Kalp Yapıları

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

fluorescent-labeling-of-drosophila-heart-structures

Research

JoVE Journal

Biology

Fluorescent Labeling of Drosophila Heart Structures

13.6K Görüntüleme.  San Diego State University. Burada larva ve yetişkin kalp tüplerin farklı unsurları floresan etiketlenmesi için temel bir protokol tanımlamak Drosophila melanogaster. Bu örnekler, floresan veya konfokal mikroskopi ile görüntüleme için çok uygundur. Bu teknik, güçlü bir model organizma kalplerin özellikleri ayrıntılı yapısal analiz izin verir.

Video: Fluorescent Labeling of Drosophila Heart Structures

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Optik Görüntüleme ile non-invaziv Algılama için Floresan Boyalar Kök Hücreler Etiketleme

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

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 ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

Dayak Kalp görselleştirme Drosophila</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

Larvaların olarak izlenmesi Kalp Fonksiyonu Drosophila melanogaster Fizyolojik Çalışmaları

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

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 ...

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae.  In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies.  The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins.  Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

Antikor Etiketleme Drosophila politen Kromozom kabak hazırlanması

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

Canlı Hücre Görüntüleme için SNAP-tag Fusion Proteinler ifade COS-7 Floresan Etiketleme

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Tüm Dağı RNA Floresan<em&gt; In situ</emVe&gt; Hibridizasyon<em&gt; Drosophila</em&gt; Embriyolar

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Floresan Proteinler ifade Canlı Farelerde Görüntüleme hücrealtı Yapıların Intravital Mikroskopi

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

Drosophila Hazırlama ve Optik Koherens Mikroskobu Kullanımı In Vivo Kalp Fonksiyonu Boyuna Görüntüleme (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...