ونود أن نشكر ليندسي دبليو بلاسخارت وجاستن سوي على إسهاماتهما الكبيرة في التطوير المبكر لهذا البروتوكول. المؤلفون ممتنون لمجتمع الذباب على كرمهم مع الكواشف ، وخاصة لروث ليمان وبنيامين لين على هديتهم لخط nos 5'-Lifeact-tdtomato p2a tdkatushka2 Caax nos3' قبل نشره. تم استخدام المخزونات التي تم الحصول عليها من مركز بلومنغتون لذبابة الفاكهة (NIH P40OD018537) في هذه الدراسة. تم دعم هذا العمل من قبل NIH RO1 GM060804 و R33AG04791503 و R35GM136270 (S.D) بالإضافة إلى منح التدريب T32GM007229 (B.W.) و F32GM125123 (L.A).

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+live+imaging+of+stem+cells+in+drosophila+testis.">Time-lapse live imaging of stem cells in drosophila testis.</a> Current Protocols in Stem Cell Biology. 0331, 1-9 (2009).</li><li>Anllo, L., Plasschaert, L. W., Sui, J., DiNardo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+reveals+hub+cell+assembly+and+compaction+dynamics+during+morphogenesis+of+the+Drosophila+testis+niche.">Live imaging reveals hub cell assembly and compaction dynamics during morphogenesis of the Drosophila testis niche.</a> Developmental Biology. 446, 102-118 (2019).</li><li>Rebecca Sheng, X., Matunis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+the+Drosophila+spermatogonial+stem+cell+niche+reveals+novel+mechanisms+regulating+germline+stem+cell+output.">Live imaging of the Drosophila spermatogonial stem cell niche reveals novel mechanisms regulating germline stem cell output.</a> Development. 138, 3367-3376 (2011).</li><li>Aboim, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developpement+embryonnaire+et+post-embryonnaire+des+gonades+normales+et+agametiques+de+Drosophila+melanogaster.">Developpement embryonnaire et post-embryonnaire des gonades normales et agametiques de Drosophila melanogaster.</a> Revue Suisse De Zoologie. 52, 53 (1945).</li><li>Sano, H., et al. <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+actin+regulator+ENABLED+regulates+cell+shape+and+orientation+during+gonad+morphogenesis.">The Drosophila actin regulator ENABLED regulates cell shape and orientation during gonad morphogenesis.</a> PLoS One. 7, (2012).</li><li>Clark, I. B. N., Jarman, A. P., Finnegan, D. 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ليس لدى المؤلفين ما يكشفون عنه.

أثناء تكوين الغدد التناسلية ، يخضع الغدد التناسلية الجنينية ، وخاصة مكانة الخلايا الجذعية داخل الغدد التناسلية الذكرية15 ، لتغيرات مورفولوجية سريعة. يتم فهم الآليات التنموية التي تكمن وراء هذه التغييرات الديناميكية بشكل أفضل من خلال تقنيات التصوير الحي. ومع ذلك ، في المرحلة ...

وقد خدم الخصية ذبابة الفاكهة melanogaster كنموذج لفهمنا للعديد من العمليات الخلوية الديناميكية. وقد ألقت الدراسات التي أجريت على هذا النموذج الضوء على تنظيم انقسام الخلايا الجذعية1،2،3 ، وتطوير الخلايا الجرثومية4،5 ، وبيولوجيا piRNA6،7،8 ، وأحداث إشارات الخلايا الجذعية المتخصصة9،10،11،12،13. هذا النموذج مفيد لأنه قابل للسحب وراثيا14,15 وهو واحد من القلائل الذين يمكننا من خلالها تصوير الخلايا الجذعية الحية في بيئتهم الطبيعية3,16,17,18. ومع ذلك ، فقد اقتصر التصوير الحي لهذا النموذج على الأنسجة البالغة والمراحل الجنينية المبكرة ، مما ترك فجوة في معرفتنا بديناميكيات الغدد التناسلية في الجنين المتأخر ، وهي المرحلة الدقيقة التي يتشكل فيها المكان لأول مرة ويبدأ في العمل.
الغدد التناسلية الجنينية في المرحلة المتأخرة هي كرة ، تتكون من خلايا متخصصة جسدية في الأمام ، وخلايا جرثومية تنشرها خلايا الغدد التناسلية الجسدية في جميع أنحاء المناطق الخلفية19. يمكن تصوير هذا العضو حيا في الجسم الحي حتى المرحلة الجنينية المبكرة 1717،20،21. يتم منع المزيد من التصوير بسبب بدء تقلصات العضلات على نطاق واسع. هذه الانقباضات شديدة لدرجة أنها تدفع الغدد التناسلية خارج إطار التصوير ، ولا يمكن تصحيح هذه الحركة باستخدام برنامج التصوير. يهتم مختبرنا بالكشف عن آليات التكوين المتخصصة ، والتي تحدث خلال هذه الفترة المراوغة للتصوير الحي. لذلك ، أنشأنا نهجا خارج الجسم الحي للتصوير الحي للغدد التناسلية بدءا من المرحلة الجنينية 16 ، مما يسهل دراسة ديناميكيات الخلية خلال هذه الفترة الحاسمة من تطور الغدد التناسلية. يظهر العمل السابق من مختبرنا أن هذا التصوير خارج الجسم الحي يلخص بأمانة في تطوير الغدد التناسلية في الجسم الحي 17. هذه التقنية هي الأولى والوحيدة من نوعها للغدد التناسلية الجنينية ذبابة الفاكهة .
هنا ، نقدم بروتوكول التشريح المطلوب للتصوير الحي خارج الجسم الحي للغدد التناسلية خلال المراحل الجنينية المتأخرة. يمكن دمج هذا البروتوكول مع العلاجات الدوائية ، أو التلاعب المعدل وراثيا لسلالات خلايا محددة داخل الغدد التناسلية. باستخدام هذه التقنية ، نجحنا في تصوير خطوات تكوين الخلايا الجذعية المتخصصة17. وبالتالي ، فإن نهج التصوير هذا مفيد في مجال بيولوجيا الخلايا الجذعية ، لأنه سيمكن من تصور المراحل الأولية من تكوين المتخصصة في الوقت الفعلي داخل بيئتها الطبيعية15,17. في حين أن هذه الطريقة مفيدة لمجال بيولوجيا الخلايا الجذعية ، إلا أنها قابلة للتطبيق أيضا لتصور أي عمليات ديناميكية تحدث في الغدد التناسلية خلال هذه النقطة الزمنية التنموية ، بما في ذلك إعادة الترتيب الخلوي 22 ، والتصاق الخلايا2،12،23 ، وهجرة الخلايا 23. وبالتالي فإن بروتوكول التشريح هذا سيعزز فهمنا للعديد من العمليات البيولوجية الأساسية للخلايا.

<table>
	<tbody>
		<tr>
			<td>Alfa Aesar&#160;Tungsten wire</td>
			<td>Fisher Scientific</td>
			<td>AA10408G6</td>
			<td>0.25mm (0.01 in.) dia., 99.95% (metals basis)</td>
		</tr>
		<tr>
			<td>D. melanogaster: His2Av::mRFP1</td>
			<td>Bloomington Drosophila Stock Center (BDSC)</td>
			<td>FBtp0056035</td>
			<td>Schuh, Lehner, &#38; Heidmann, Current Biology, 2007</td>
		</tr>
		<tr>
			<td>D. melanogaster: nos-lifeact::tdtomato</td>
			<td>Gift from Ruth Lehmann Lab</td>
			<td></td>
			<td>Lin, Luo, &#38; Lehmann, Nature Communications, 2020: nos5&#39;- Lifeact-tdtomato p2a tdkatushka2 Caax nos3&#39;</td>
		</tr>
		<tr>
			<td>D. melanogaster: P-Dsix4-eGFP::Moesin</td>
			<td></td>
			<td>FBtp0083398</td>
			<td>Sano et al., PLoS One, 2012</td>
		</tr>
		<tr>
			<td>Diamond-tipped knife</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Double-sided tape</td>
			<td>Scotch</td>
			<td>665</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>GIBCO</td>
			<td>10082</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging dish</td>
			<td>MatTek</td>
			<td>P35GC-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging software</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>MetaMorph Microscopy Automation and Image Analysis Software v7.8.4.0</td>
		</tr>
		<tr>
			<td>Insulin, bovine</td>
			<td>Sigma</td>
			<td>l0516</td>
			<td>Store aliquots at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Fisher Scientific</td>
			<td>08-955</td>
			<td></td>
		</tr>
		<tr>
			<td>Nytex basket</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Corning</td>
			<td>30-002-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>Ringer&#39;s solution</td>
			<td></td>
			<td></td>
			<td>2 mM MgCl2, 2 mM CaCl2, 130 mM NaCl, 5mM KCl, 36 mM Sucrose, 5mM Hepe&#8217;s Buffer; adjusted with NaOH until pH of 7.3 is achieved</td>
		</tr>
		<tr>
			<td>Schneider&#39;s Insect Media</td>
			<td>GIBCO</td>
			<td>21720-024</td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection and live-imaging of the late embryonic drosophila gonad

يعد الغدد التناسلية الجنينية الذكرية ذبابة الفاكهة نموذجا مفيدا لدراسة الجوانب المختلفة لعلم الأحياء التنموي بما في ذلك ، على سبيل المثال لا الحصر ، تطور الخلايا الجرثومية ، وبيولوجيا piRNA ، وتكوين المتخصصة. هنا ، نقدم تقنية تشريح للتصوير الحي للغدد التناسلية خارج الجسم الحي خلال فترة يكون فيها التصوير الحي في الجسم الحي غير فعال للغاية. يحدد هذا البروتوكول كيفية نقل الأجنة إلى طبق تصوير، واختيار الأجنة الذكرية ذات المراحل المناسبة، وتشريح الغدد التناسلية من الأنسجة المحيطة بها مع الحفاظ على سلامتها الهيكلية. بعد التشريح ، يمكن تصوير الغدد التناسلية باستخدام مجهر متحد البؤرة لتصور العمليات الخلوية الديناميكية. يتطلب إجراء التشريح توقيتا دقيقا وبراعة ، لكننا نقدم نظرة ثاقبة حول كيفية منع الأخطاء الشائعة وكيفية التغلب على هذه التحديات. على حد علمنا ، هذا هو أول بروتوكول تشريح للغدد التناسلية الجنينية ذبابة الفاكهة ، وسوف يسمح بالتصوير الحي خلال نافذة زمنية يتعذر الوصول إليها بخلاف ذلك. يمكن دمج هذه التقنية مع التلاعب الدوائي أو الخلوي المحدد المعدل وراثيا لدراسة أي عمليات ديناميكية تحدث داخل أو بين الخلايا في بيئتها التناسلية الطبيعية.

نوضح إعداد طبق التصوير في الشكل 1 ، كما هو موضح في "إعداد يوم التشريح". يجب أن تؤدي هذه الطرق في النهاية إلى التصاق أجنة رطبة جيدا بشريط انزلاق الغطاء ، والذي يتم تثبيته مؤقتا في قاع الطبق وغمره في محلول رينجر (الشكل 1F). تسمح السكين ذات الرؤوس الماسية للمرء بتق?...

Watch this Scientific Journal Video about Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad at JoVE.com

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

هنا ، نقدم بروتوكول تشريح مطلوب لتصوير الغدد التناسلية الذكرية ذبابة الفاكهة الجنينية المتأخرة . سيسمح هذا البروتوكول بمراقبة العمليات الخلوية الديناميكية في ظل الظروف العادية أو بعد التلاعب المعدل وراثيا أو الدوائي.

تشريح وتصوير حي للغدد التناسلية ذبابة الفاكهة الجنينية المتأخرة

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, ...

dissection-and-live-imaging-of-the-late-embryonic-drosophila-gonad

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

3.1K Views.  University of Pennsylvania. هنا ، نقدم بروتوكول تشريح مطلوب لتصوير الغدد التناسلية الذكرية ذبابة الفاكهة الجنينية المتأخرة . سيسمح هذا البروتوكول بمراقبة العمليات الخلوية الديناميكية في ظل الظروف العادية أو بعد التلاعب المعدل وراثيا أو الدوائي.

Video: Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific mRNA.

على نطاق واسع اسماك الزرد الجنينية القلب تشريح لتحليل الترانسكربتي

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

يعيش التصوير لل<em&gt; ذبابة الفاكهة</em&gt; العذراء العين

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

تشريح وثقافة ماوس الجنينية الكلى

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

Long-term Live Imaging of Drosophila Eye Disc

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have been used to study many biological processes, such as cell proliferation, differentiation, growth, migration, apoptosis, competition, cell-cell signaling, and compartmental boundary formation. However, methods for the long-term ex vivo culture and live imaging of the imaginal discs have not been satisfactory, despite many efforts. Recently, we developed a method for the long-term ex vivo culture and live imaging of imaginal discs for up to 18 h. In addition to using a high insulin concentration in the culture medium, a low-melting agarose was also used to embed the disc to prevent it from drifting during the imaging period. This report uses the eye-antennal discs as an example. Photoreceptor R3/4-specific m&#948;0.5-Ga4 expression was followed to demonstrate that photoreceptor differentiation and ommatidial rotation can be observed during a 10 h live imaging period. This is a detailed protocol describing this simple method.

Long-term Live Imaging of Drosophila Eye Disc

This protocol describes a detailed method for the long-term ex vivo culture and live imaging of a Drosophila imaginal disc. It demonstrates photoreceptor differentiation and ommatidial rotation within the 10 h period of live imaging of the eye disc. The protocol is simple and does not require expensive setup.

طويلة الأجل تصوير لايف<em&gt; ذبابة الفاكهة</em&gt; القرص العين

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

الهندسة الحيوية من أنسنة نخاع العظام ميكرونفيرونمنتس في الماوس والتصور من خلال التصوير لايف

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

تصوير لايف من ماوس الثانوي باليت فيوجن

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

تشريح وتلطيخ إيممونوفلوريسسينت للفطر الجسم وعلم الخلايا العصبية في أدمغة البالغين melanogaster المورفولوجية

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

تشريح وتلطيخ المبايض المورفولوجية الخوادر

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction.&#160;

تشريح الشبكية الخوادر المورفولوجية إيمونوهيستوتشيميستري والتحليل الغربي، وعزل الحمض النووي الريبي

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Here, we present a method to record embryonic muscle contractions in Drosophila embryos in a non-invasive and detail-oriented manner.

التصوير الحي وتحليل تقلصات العضلات في جنين دروسوفيلا

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...