Sohbet Marie Curie yeniden bütünleşme hibe ile desteklenmektedir. Bu çalışma, İsrail Bilim Fonu Hibe No ile desteklenmiştir. 1146-1108, Helen ve Weizmann Bilim Enstitüsü Kök Hücre Araştırma ve Leir Vakfı Martin Kimmel Enstitüsü.

1. Döşeme Yumurta <ol><li> Diseksiyonu beş gün önce: 2-4 saat evlendirilen kadın, maya ile desteklenmiş taze gıda yumurtalarını bırakmak için izin verir. Senkronize ve iyi gelişmiş larva almak önemlidir kalabalık değil kültürlü (yaklaşık 30 yumurta / 25mm flakon). Tipik olarak, ne kadar iyi onlar yatıyordu bağlı olarak 7-16 kadın, şişe başına kullanılır. </li></ol> 2. Larvaları seçilmesi <ol><li> Ringer orta (128mm NaCl, 2mm KCl, 1.8mm CaCl 2, 4mm MgCl 2, 35.5mM Sakkaroz, 5mM HEPES pH 6.9) ile dolu bir 9-iyi cam diseksiyon tabak hazırlayın. </li><li> Ringer orta ve buz üzerine yerleştirin içeren altı plaka hücre süzgeçler hazırlayın. Alternatif olarak, naylon örgü ile donatılmış özel yapılmış kalıplar kullanın. </li><li> Flakon duvarları ince biyolojik cımbızla (forseps) kullanarak zaman larvaların seçin ve ringer içeren diseksiyon çanak koyun. </li><li> Temiz bir iyi bir kadın larva transferi. Biz kendi gonadlar tarafından erkek kadın ayırt eder. Testislere kolayca yağ vücudun posterior üçte gömülü büyük açık oval olarak tanımlanır. Kadın yumurtalıklar, yağ vücudun aynı kısmında bulunan çok daha küçük, açık, yuvarlak bir küre olarak tespit edilebilir. </li></ol> 3. Larva diseksiyonu <ol><li> Forseps ile beyin sadece arka larva tutun ve ikinci bir çift forseps ile baş kaldırmak. </li><li> Trakea aşağı bakacak şekilde, sırt tarafında kalan arka kısmı yerleştirin. </li><li> Forseps bir çift ile arka spiracles larva tutun ve diğer çifti, yaklaşık yarım larva yağ vücut çıkıncaya kadar posteriora manikür sürgülü ise içe doğru yavaşça itin. </li><li> Posterior ucunu sıkıca tutun ve manikür gevşek tutmak için forseps diğer çifti kullanın. Posterior uç ve yavaşça çekmeye aralağından manikür ve bağlı bağırsak slayt böylece. Bu sürecin sonunda, bağırsak ve manikür yağ vücut tamamen ayrılmış olmalıdır. Bağırsakta yağ vücudun ön kısmından ayırın. Boyama ve montaj kolaylaştırmak için, yağ vücut sağlam kalacağını önemlidir. </li><li> Tercihen de içeren disseke larvaları, Ringer orta mera pipet ile iyice ıslatın. Bu katın pipet yardımcı olur ve yağ vücuda yapışmasını önler. Yağlı vücut hücre süzgeç buz Ringer ortama aktarmak için mera pipet kullanın. </li></ol> 4. Fiksasyon ve Boyama Tüm adımlar 4, ilk antikor ile inkübasyon dışında, oda sıcaklığında yapılır ° C <ol><li> Ringer orta% 5 Formaldehit yağ vücut inkübe edin. 20 dakika boyunca yavaşça sallanarak. </li><li> % 1 PBT (% 1 Triton PBS içinde x-100) ile 5 dakika yıkayınız. 10 dakika ve 45 dakika için üçüncü kez bu adımı tekrarlayın. % 1 Triton X-100 larva yumurtalık perfore ve antikorlar nüfuz izin gereklidir. </li><li> Yavaşça sallanarak ile 1 saat boyunca,% 0.3 PBTB (% 0.3 Triton X-100 ve PBS içinde% 1 BSA) Blok </li><li> 4 gece boyunca% 0.3 PBTB seyreltilmiş istenen 1. antikor ° C yavaşça sallanarak ile inkübe edin. Bu adım, genellikle bir rulo 0.2 ml tüpler yapılır. </li><li> Yağ vücut hücre süzgeçler geri aktarın. </li><li> Yavaşça sallanarak ile% 0.3 PBTB ile 3 kez, her biri 30 dakika yıkayın. </li><li> % 0.3 PBTB yavaşça sallanarak ile 1 saat süreyle% 5 normal eşek serum ile takviye ile engelleyin. </li><li> (Üreticisinin teknik verilerine göre) ile 2 saat boyunca yavaşça sallanarak engelleme solüsyonu (% 0.3,% 0.3 PBT% 5 normal eşek serum ile desteklenmiş PBTB) seyreltilmiş uygun bir ikincil antikor ile inkübe edin. Antikor floresan ise, bu noktadan itibaren karanlıkta inkübe edin. Bu adım, genellikle bir rulo 0.2 ml tüpler yapılır. </li><li> 3 kez her yavaşça sallanarak% 0.3 PBT ile 30 dakika yıkayın. </li></ol> 5. Montaj <ol><li> Yağlı vücut temiz Eppendorf tüpüne aktarın. Çok dikkatli bir şekilde bütün sıvılar kaldırmak ve hemen Vectashield montaj medya ile kaplayın. Yumurtalıkların yağ vücuttan ayrılmış edilirken sertleşme olmadığından rutin vectashield kullanın. Bir hafta için numuneler 4 ° C&#39;de montaj medya saklanabilir. </li><li> Bir pipet ucu sonunda kesin ve dikkatli bir mikroskop lamı üzerine montaj medya minimal volüm (30mm kapak kayma için 30 ul) ile yağlı vücut aktarmak için kullanabilirsiniz. </li><li> Yağlı vücut yaymak için 0,1 mm çapında pimleri tutarak, iki nikel kaplama pin sahipleri kullanın. Yumurtalıklar yağ vücudun posterior üçte yer almaktadır. Bunları çevreleyen yağ vücut genellikle bir &#39;çiçek&#39; şeklindedir. Bu yumurtalıkların yerini belirlemenize yardımcı olur. Yağ vücudun geri kalanına &#39;çiçekler&#39; inceleyin ve ikincisi atın. </li><li> Yağlı vücut surro çıkarındikkatli bir şekilde iki iğne etrafında geçerek gonad unding. Yağlı vücut kalıntı uzak slayt izole gonad yerleştirin. </li><li> Oje ile bir kapak kayma ve mühür ile kaplayın. </li><li> Konfokal bir mikroskop kullanılarak doğrudan gözünüzde canlandırın. Numuneler üç hafta kadar 4 ° C&#39;de muhafaza edilebilir. </li></ol> 6. Temsilcisi Sonuçlar Lerin ve onların somatik nişler da dahil olmak üzere, somatik yumurtalık içinde birkaç hücre soyları, kurulmasını takip için yukarıdaki protokolünü kullandık. Bu amaçla, gelişmekte olan gonad farklı hücre tipleri ayırt etmek için özel antikorlar ve işaretleri kullanabilirsiniz. Burada antikorların farklı kombinasyonları ile boyanmış iki LL3 yumurtalıkların bir örnek göstermektedir. Şekil 1A birlikte niş somatik hücre oluşturan terminal filament ve kap hücreleri (yeşil), vurgulamaktadır. Şekil 1B, germ hücreleri (mavi) doğrudan temas Puntolu hücreler (IC, macenta) gösterir. <img src="/files/ftp_upload/2537/2537fig1.jpg" alt="Şekil 1" /> Şekil 1 LL3 yumurtalıklar. (A) monoklonal antikor 1B1 (magenta), somatik hücre ve lekeleri fusome PGCs içinde bir hücre içi organel (oklar) özetlenir. Arttırıcı tuzak ss-lacZ (anti-β-galaktosidaz, yeşil) terminali filamentler olarak ifade edilir. Tabanda filament somatik kap hücreleri (ok başları) görülebilir. (B) 1B1 antikor (yeşil), yumurtalık tüm somatik hücre özetliyor. Anti-Vasa (mavi), tüm PGCs etiketler. PGCs doğrudan Puntolu hücreler (IC, anti-trafik sıkışıklığı, kırmızı) başvurun. Bar (A ve B için) 20 mikron.

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Bu video geç üçüncü instar larva yumurtalıkların bir izolasyon ve boyama protokolü göstermektedir. Bu protokol rutin ve güvenilir yapmak için, dikkat, aşağıdaki noktalara dikkat edilmelidir: <ol><li> Senkronize ve iyi gelişmiş larvalar için, üzerinde kalabalık kaçınılmalıdır. </li><li> Küçük saydam yumurtalıkların kaybını önlemek için, yağ vücut sağlam incelemek için emin olun. Bu aynı zamanda, özellikle (ikinci ya da üçüncü instar başında) genç yumurtalıklar montaj, mo...

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<td>NaCl</td>
<td>&nbsp;</td>
<td>J.T.Baker</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Kcl</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>CaCl2</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>MgCl2</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Sucrose</td>
<td>&nbsp;</td>
<td>J.T.Baker</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Hepes</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>PBS</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
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<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Albumin Bovine Fraction V</td>
<td>&nbsp;</td>
<td>MP Biomedicals</td>
<td>160069</td>
<td>&nbsp;</td>
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<td>Dumont biology tweezers 5 dumstar polished</td>
<td>&nbsp;</td>
<td>FST Fine Science Tools</td>
<td>11295-10</td>
<td>&nbsp;</td>
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<td>Nickel plated pin holder</td>
<td>&nbsp;</td>
<td>FST Fine Science Tools</td>
<td>26018-17</td>
<td>&nbsp;</td>
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<td>s.s minutien pins 0.1mm diam, 10mm long</td>
<td>&nbsp;</td>
<td>FST Fine Science Tools</td>
<td>26002-10</td>
<td>&nbsp;</td>
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<td>9 well plates 85X100 mm, 22mm o.d.x7mm deep</td>
<td>&nbsp;</td>
<td>CORNING</td>
<td>7220-85</td>
<td>&nbsp;</td>
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<td>Stereo Microscope MZ 16.5 with a standard transmitted light base TL ST</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>&nbsp;</td>
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<td>6 well plates</td>
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<td>Costar</td>
<td>3516</td>
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<td>Slides</td>
<td>&nbsp;</td>
<td>Menzel Glaser GmbH</td>
<td>798</td>
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<td>Cover slips</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>2940-223</td>
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<td>Mounting media</td>
<td>&nbsp;</td>
<td>Vectashield</td>
<td>H-1200</td>
<td>&nbsp;</td>
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<td>Cell strainer</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>FAL352350</td>
<td>&nbsp;</td>
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<td>1B1 antibody</td>
<td>&nbsp;</td>
<td>Developmental Studies Hybridoma Bank</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Anti-Traffic Jam</td>
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<td>Laboratory of Dr. Dorothea Godt</td>
<td>&nbsp;</td>
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<td>Anti-Vasa</td>
<td>&nbsp;</td>
<td>Laboratory of Dr. Ruth Lehmann</td>
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<td>Anti &beta;-Galactosidase</td>
<td>&nbsp;</td>
<td>Cappel</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Secondary Antibodies</td>
<td>&nbsp;</td>
<td>Jackson ImmunoResearch</td>
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dissection and staining of drosophila larval ovaries

Birçok organların embriyogenez sırasında ve yetişkin ömrü boyunca bakım ve onarım için onların gelişimi için kök hücreleri bağlıdır. Onların çevre ile etkileşim kök hücre nasıl form ve nasıl olduğunu anlamak geliştirme, homeostaz ve hastalığı anlamak için bu nedenle çok önemlidir. Meyve sineği Drosophila melanogaster yumurtalık somatik destek hücreleri (niş) 1, 2 germ line kök hücreler (lerin) etkileşim için etkili bir model olarak görev yaptı. Genetik olarak onları manipüle etme yeteneği ile birleştiğinde, niş ve lerin bilinen yerde, araştırmacılar kök hücreleri ve nişler 3-12 arasındaki etkileşimleri çeşitli aydınlatmak için izin verdi. GSC bakım ve farklılaşma kontrol mekanizmaları hakkında bilgi zenginliği rağmen, nispeten daha az lerin ve somatik nişler gelişimi sırasında formu hakkında bilinmektedir. Olan hücresel bileşenleri terminal filament ve kap hücreleri (Şekil 1), 13-17 sırasında instar üçüncü larva formu somatik nişler, yaklaşık 18. Lerin primordial germ hücrelerinin (PGCs) kaynaklanır. PGCs erken larva aşamasında çoğalırlar, ancak niş oluşumu takip PGCs bir alt lerin 7, 16, 18, ​​19 olur. Birlikte, somatik niş hücre ve lerin organizmanın ömrü boyunca yumurta üreten işlevsel bir birim. GSC birim oluşumu ile ilgili birçok soru cevapsız kalmaktadır. Prekürsör hücreler, nişler ve kök hücre öncülleri veya lerin haline PGCs içinde asimetri nesil arasında koordinasyon gibi işlemler en iyi larva incelenebilir. Bununla birlikte, larva yumurtalık gelişme metodik bir çalışma, fiziksel olarak zordur. Birincisi, larva, yumurtalıklar küçük. Geç larva aşamasında bile onlar arasında sadece 100μm. Buna ek olarak, yumurtalıklar, şeffaf ve beyaz yağ vücut gömülü. Burada floresan antikorlar ile boyanarak takip geç üçüncü instar (LL3) Drosophila larvaları, yumurtalıklar izole etmek için bir adım-adım protokolünü açıklar. Biz, gibi sorunlar, yumurtalıkların yerini, lavabo değil doku boyama ve yıkama ve antikorlar doku içine nüfuz emin olun bazı teknik çözümler sunuyoruz. Bu protokol, daha önce larva aşamaları ve larva testisler gibi uygulanabilir.

Watch this Scientific Journal Video about Dissection and Staining of Drosophila Larval Ovaries at JoVE.com

Dissection and Staining of Drosophila Larval Ovaries

Nişler ve kök hücre gelişimi sırasında formu nasıl pratik etkileri olan önemli bir soru. Giriş<em&gt; Drosophila</em&gt; Yumurtalık, germ line kök hücreleri ve somatik nişler larva gelişimi sırasında oluşturur. Bu video geç üçüncü instar (LL3) kadın gonadlar, teşrih leke ve monte etmek için nasıl gösterir<em&gt; Drosophila</em&gt; Larvaları.

Diseksiyon ve boyanması Drosophila Larvaların Yumurtalıkların

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

dissection-and-staining-of-drosophila-larval-ovaries

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Biology

Dissection and Staining of Drosophila Larval Ovaries

26.1K Görüntüleme.  Weizmann Institute of Science. Nişler ve kök hücre gelişimi sırasında formu nasıl pratik etkileri olan önemli bir soru. Giriş Drosophila Yumurtalık, germ line kök hücreleri ve somatik nişler larva gelişimi sırasında oluşturur. Bu video geç üçüncü instar (LL3) kadın gonadlar, teşrih leke ve monte etmek için nasıl gösterir Drosophila Larvaları.

Video: Dissection and Staining of Drosophila Larval Ovaries

Dissection of Drosophila Ovaries

Drosophila Yumurtalıkların diseksiyonu

Biyoloji

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

Drosophila melanogaster Larvaların MSS diseksiyonu

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

Drosophila Larvaların NMJ Diseksiyon

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

Drosophila Larvaların NMJ İmmünohistokimya

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.

VE Canlı Diseksiyon Drosophila Embriyolar: Aerodinamik Antikor Boyama Mutant Koleksiyonlar Eleme Yöntemleri

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

Larvaların Kalp Diseksiyon, Çocuk ve Yetişkin Zebra balığı, Danio rerio</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and organogenesis. Immunostaining allows for the visualization of developing tissues and organs. However, a protective cuticle that forms at the end of embryogenesis prevents permeation of antibodies into late-stage embryos and larvae. While dissection prior to immunostaining is regularly used to analyze Drosophila larval tissues, it proves inefficient for some analyses because small tissues may be difficult to locate and isolate. Sonication provides an alternative to dissection in larval Drosophila immunostaining protocols. It allows for quick, simultaneous processing of large numbers of late-stage embryos and larvae and maintains in situ morphology. After fixation in formaldehyde, a sample is sonicated. Sample is then subjected to immunostaining with antigen-specific primary antibodies and fluorescently labeled secondary antibodies to visualize target cell types and specific proteins via fluorescence microscopy. During the process of sonication, proper placement of a sonicating probe above the sample, as well as the duration and intensity of sonication, is critical. Additonal minor modifications to standard immunostaining protocols may be required for high quality stains. For antibodies with low signal to noise ratio, longer incubation times are typically necessary. As a proof of concept for this sonication-facilitated protocol, we show immunostains of three tissue types (testes, ovaries, and neural tissues) at a range of developmental stages.

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Immunostaining is an effective technique for visualizing specific cell types and proteins within tissues. By utilizing sonication, the protocol described here alleviates the need to dissect Drosophila melanogaster tissues from late-stage embryos and larvae before immunostaining. We provide an efficient methodology for the immunostaining of formaldehyde-fixed whole mount larvae.

Geç evre embriyonik ve larva soniklenmesi-kolaylaştırdı Immunoflorasan Boyama<em&gt; Drosophila</em&gt; Dokular<em&gt; Yerinde</em

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

Diseksiyon ve hayali Disklerin immün itibaren<em&gt; Drosophila melanogaster</em

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...

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 ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

Larvaların Faaliyet Ölçümü<em&gt; Drosophila</em&gt; Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...