Name: immunostaining of whole-mount drosophila testes for 3d confocal analysis of large spermatocytes
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Antikorlar için P. Verrijzer, J. Johansen H., Okhura ve sinekler için A. Debec, Bloomington ve Viyana stok merkezlerine teşekkür ederiz. Öneriler ve teşvik edici tartışmalar için C. Jackson'a teşekkürler.

1. Drosophila testis koleksiyonu
NOT: Genç erkekler (eklozyon sonrası 0-15 saat) diploid mikrop hücrelerini (spermatogonia ve spermatositler) incelemek için gereklidir.
<ol><li>Spermatidler geliştirmeden kaynaklanan paraziti en aza indirmek için mümkün olduğunca genç sinekleri seçin.</li>
	<li>Karbondioksite kısa bir süre maruz kalarak sinekleri uyuşturun ve bir sineklik pedine aktarın14,15.</li>
	<li>Bir diseksiyon mikroskobu (10x büyütme) altında, başı çıkarmak için tokmak kullanın.</li>
	<li>Siyah bir arka plan desteği üzerine silikon kaplı cam kaydırakta 5 ila 10 kafa kesilmiş sinekleri 100 μL Ringer tamponu (183 mM KCI, 47 mM NaCl, 10 mM Tris-HCI, 1 mM EDTA, komple protein inhibitörü, pH 6.8) aktarın.</li>
	<li>40x büyütmede mikroskobun parçalanmasıyla, toraks ile karın arasında bir sinek tutmak için bir çift 5 numaralı toparlama kullanın ve diğer toparlamalarla birlikte karnı torakstan çekin. Testisleri ve bitişik dokuları sinek karkası15'ten hızla izole edin ve ringer tamponunun yeni bir damlasına aynı slaytta yerleştirin.</li>
	<li>Tüm testisler hazır olduğunda, testisleri kalan dokulardan (aksesuar bezi ve dış cinsel organ) temizleyin.</li>
	<li>Parçalanmış testisleri içeren damladan fazla tamponu çıkarın. Tipik olarak, 200 μL ucun üzerine monte edilmiş 10 μL uçlu bir p200 pipet kullanın. Bu, sıvı fazlalığının küçük bir diyafram açıklığından çıkarılmasını sağlar. Parçalanmış testisleri içeren damlanın üzerine% 4 paraformaldehit fiksatif bir damla yerleştirin ve ardından testisleri hızlı bir şekilde taze fiksatif çözelti içeren bir tüpe aktarın.</li>
</ol>2. Paraformaldehit fiksasyonu
<ol><li>Kullanımdan hemen önce 2 mL fiksatif çözelti hazırlayın: 2 mL silikonlu mikrosenj tüpte 30 μL NP40% 20 artı 1200 μL hepatik ile fosfat tamponlu salin (PBS) ile seyreltilmiş% 4 paraformaldehitin 600 μL'si. 600 μL aliquots olarak hazırlanan ve -20 °C'de depolanan PBS'de %4 paraformaldehit kullanın. Dikkat: Heptan ve paraformaldehit toksiktir ve duman kaputunda ele alınmalıdır. Ev sahibi enstitünün yönetmeliklerine göre bunları atın.</li>
	<li>Testleri geniş diyafram ucuna sahip bir p200 mikropipette kullanarak PFA/NP40/heptane çözeltisine aktarın. Tüpü elle 30 sn boyunca yukarı ve aşağı sallayın. Testisleri oda sıcaklığında 30 dakika boyunca döner karıştırıcıya sabitleye devam edin.</li>
	<li>Döner karıştırıcıyı durdurun ve faz ayrımına izin vermek ve testislerin tüpün dibine yerleşmesini sağlamak için tüpü bir tüp rafı içine yerleştirin.</li>
	<li>Tüm heptane ve fiksatif çözeltiyi çıkarın ve testisleri 1x PBS ile üç kez hızlı bir şekilde durulayın.</li>
	<li>Temiz bir 200 μL bireysel tüpe aktarın. Kullanılan antikor miktarlarını en aza indirmek için bu küçük tüplerdeki tüm inkübasyonları gerçekleştirin.</li>
	<li>Hem P200 hem de P10 ucu takılı 200 μL mikropipette kullanarak fazla tamponu çıkarın. Testisleri aspire etmemeye dikkat edin.</li>
	<li>Tüm numuneler hazır olana kadar testisleri buz üzerinde 1x PBS'de tutun.</li>
</ol>3. Çözeltide antikor lekesi
<ol><li>PBS'yi atın ve hücre zarlarını permeabilize etmek için oda sıcaklığında 30 dakika boyunca testisleri% 0,3 PBS-T'de bırakın.</li>
	<li>Aynı tamponda önceden inkübasyon artı 1 saat boyunca% 5 normal keçi serumu (NGS).</li>
	<li>%0,3 PBS-T artı %5 NGS(Malzeme Tablosu)ile seyreltilmiş birincil antikorun 200 μL'sini ekleyin.</li>
	<li>Birincil antikoru bir gecede 4 °C'de (veya oda sıcaklığında) bir rocker üzerinde hafif ajitasyonla kuluçkaya yatırın.</li>
	<li>Oda sıcaklığında bir rocker üzerinde 15 dakika boyunca% 0.3 PBS-T'de 3 kez yıkayın. Testislerin yerçekimi ile tüpün dibine yerleşmesine izin verin.</li>
	<li>DyLight konjuge serisinden 1:500 seyreltilmiş 200 μL ikincil antikor ekleyin.</li>
	<li>İkincil antikorun oda sıcaklığında 4 saat boyunca karanlık bir odada kuluçkaya yatırın.</li>
	<li>Oda sıcaklığında bir rocker üzerinde 15 dakika boyunca% 0.3 PBS-T'de yıkayın. Daha sonra her seferinde oda sıcaklığında bir rocker üzerinde 30 dakika boyunca% 0.2 PBS-T ve% 0.1 PBS-T ile tekrarlayın. Deterjanı çıkarmak için 30 dakika boyunca 1x PBS'de son bir yıkama yapın.</li>
	<li>15 dakika boyunca 1x PBS'de 1 μg/mL'de 180 μL DAPI çözeltisi ekleyin. Stok çözeltisi H2O'da 10 mg/mL'dir. PBS'de stok çözeltisi yapmaktan kaçının, bu da daha büyük spermatositlerde dağınık kromatin üzerindeki DAPI boyama sinyalini azaltabilir.</li>
	<li>Her biri 5 dakika boyunca 3x yıkayın.</li>
	<li>Temiz bir slayt üzerinde bir daire çizmek için hidrofobik bir bariyer kalemi kullanın.</li>
	<li>Testisleri aspire edin ve slayda koyun. Geniş diyafram ucunun %0,1 PBS-T ile önceden durulanmasını test etmenin uyrmaya yapışmasını önler. Fazla PBS'yi kaldırın.</li>
	<li>Citifluor AF1'den bir damla (yaklaşık 100 μL) ekleyin.</li>
	<li>Hava kabarcıklarını hapsetmekten kaçınmaya dikkat ederek, dokunun üzerine hafifçe bir cam kapak yerleştirin.</li>
	<li>Kapak kapağını şeffaf oje kullanarak kaydırağa kapatın.</li>
	<li>Slaytları mikroskopta gözlemleyin.</li>
</ol>4. Kolokalizasyon analizleri
<ol><li>Konfokal görüntüler elde edin. Z çözünürlüğünde 0,5 veya 1 μm çözünürlüğünde zeiss LSM 710 konfokal mikroskop (63x Plan Apokromatik yağ) kullandık.</li>
	<li>Bağımsız testislerden farklı hücreler seçin.</li>
	<li>Görüntüleri işleme yazılımına (örneğin, imaris) içe aktarın.</li>
	<li>Komşu çekirdeği dışlamak için çekirdeği manuel segmentasyonla tanımlayın. Coloc yazılımını kullanın; Pearson'ın korelasyon katsayılarını (PCC' ler) 2 florofor arasındaki tüm hacimde toplar.</li>
</ol>

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Yazarların açıklayacak bir şeyi yok.

Bu protokol, tüm mount yetişkin Drosophila testislerinin başarılı bir şekilde immünoblabeling için bir yöntem sağlar. Diğer prosedürlerde bildirildiği gibi, spermatositleri engelleme riski taşıyan spermin varlığını en aza indirmek için genç erkeklerin kullanılması gerekir (hatta eklosyondan sonra yaşı 20 saatten daha az bir süreye kısalttık). Diseksiyon hızlı olmalı ve antikor seyreltmesi sinyal-gürültü oranını optimize etmek için ayarlanmalıdır21,...

Drosophila testisleri nükleer mimarinin incelenmesi için değerli bir modeldir. Mitotik spermatogonyal hücrelerde, nükleer hacim büyük ölçüde kromatin tarafından işgal edilir. Bu hücreler dört tur bölünmeye uğrar ve 16 birincil spermatosit kisti üretir. Sonraki birkaç gün boyunca, spermatositler 6 gelişim aşamasından (S1-S6) geçer, hacim olarak büyük ölçüde artar, yaklaşık olarak 20 kat1,2. Nükleer morfolojilerini de değiştiriyorlar. Lamin Dm0 tarafından ortaya çıkan çekirdeğin ana hatları düzensiz hale gelir ve derin invaginasyonları gösterir1,3. Buna kromatin organizasyonu 1 , 4 ,5,6,7 'de kapsamlı gen ekspresyasyonu değişiklikleri ve modifikasyonları eşlikeder. Ara evre kromozomları, S4-S5 evresinde iyi tanımlanmıştır ve 2 büyük çift değerli otomoz ve çekirdekle sabitlenmiş X-Y çiftine karşılık gelen üç ayrı kütle oluşturur.
Mad1 başlangıçta mitotik kontrol noktasının önemli bir bileşeni olarak izole edildi(8'degözden geçirildi). Interfazda, öncelikle nükleer zarfta değil, aynı zamanda nükleoplazma9, 10,11'dede bulunur. Drosophila testislerinde, Mad1'in nükleoplazmada belirgin olduğunu, iki büyük otozomal kromatin kütlesi ile yakından ilişkili olduğunu ve XY kromatin ile daha az ölçüde ilişkili olduğunu bildirdik. Kromatinle ilişkili bu yapıyı MINT (Mad1 içeren IntraNükleer Bölge)12olarak tanımladık. Diğer dört protein spermatositlerdeki MINT'lerle ilişkiliydi: nükleoporin Mtor / Tpr, SUMO peptidaz Ulp1, mitotik kontrol noktası proteini Mad2 ve Polikomla benzeri bir kompleksin alt bireyi Raf212.
MINT'lerin tanımını tamamlamak için antikor kullanmamız gerekiyordu ve testislerde genellikle immünostaining ile karşılaşılan teknik sorunlarla karşı karşıya kaldık: testislerin derinliğinde, özellikle de büyük spermatositlerde önemli bir homojenlik ile sonuçlanan zayıf bir geçirgenlik. Squashed preparatları antikor erişimini artırdı, ancak sıkıştırılmış görüntülere yol açtı, 3D analizleri kısıtladı ve kolokalizasyon da dahil olmak üzerenicel floresan ölçümünü önledi. Antikor penetrasyonu sorunu da % 0.3 Na deoksikollat13gibi permeabilizasyon edici ajanlar tarafından ele alınabiliyordu, ancak bu prosedür elimizde tekrarlanabilir sonuçlar vermedi. Birçok ortak etiketleme deneyi yaptığımız için, permeabilizasyon protokolünü geliştirmeye baktık. % 1 NP40 artı heptan kombinasyonu, özellikle büyük spermatositlerin incelenmesinde daha fazla tekrarlanabilirlik ile sonuçlandı.
Bu protokol, süspansiyondaki testislerin sabitlenmesi ve immünostainingini gerçekleştirir, numune kalitesini artırır, tekrarlanabilirliği artırır ve konfokal mikroskopi için uygun hale getirir. Protokolü, bazı nükleer zarf proteinlerinin büyük spermatositlerin nükleoplazmik yapılarıyla olan mekansal ilişkilerini daha iyi tanımlamak için kullandık. Bu yöntem, spermatosit gelişimine eşlik eden değişikliklerin, örneğin kromatin, nükleoplazma ve nükleer gözenekler de dahil olmak üzere çekirdeğin dinamik yapısal değişikliklerinin daha iyi araştırılmasına izin verecektir.

<table><tbody><tr><td>&lt;em&gt;&lt;strong&gt;Drosophila srains&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>ketelGFP y; ketelGFP/y+CyO</td><td></td><td></td><td>Gift from A. Debec (Institut Jacques Monod, UMR 7592, Paris, France)</td></tr><tr><td>Mad1-GFP</td><td></td><td></td><td>J Cell Sci. 2011 May 15;124(Pt 10):1664-71.</td></tr><tr><td>Mtor RNAi&amp;nbsp;</td><td>VDRC</td><td>ID 110218</td><td>Vienna Drosophila RNAi Center</td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DAPI&amp;nbsp;</td><td>Thermo</td><td>D1306</td><td>Stock solution must be prepared in H2O</td></tr><tr><td>Dumont # 5 Fine Forseps&amp;nbsp;</td><td>Fine Science Tools</td><td></td><td></td></tr><tr><td>MBP Wide Bore Pipette Tips</td><td>Fisherbrand</td><td>11917744</td><td>Wide aperture tip</td></tr><tr><td>Thermo Scientific&amp;nbsp;0.2 mL Individual Tube</td><td>Thermo</td><td>AB-0620</td><td></td></tr><tr><td>2 ml micro centrifuge tubes&amp;nbsp;</td><td>Sigma</td><td>T3531-200EA&amp;nbsp;</td><td>Siliconized 2ml tube</td></tr><tr><td>Citifluor AF1</td><td>Citifluor</td><td>AF1</td><td></td></tr><tr><td>Dakopen&amp;nbsp;</td><td>Sigma</td><td>Z377821-1EA&amp;nbsp;</td><td></td></tr><tr><td>Normal Goat Serum (NGS)</td><td>Sigma</td><td>NS02L-1ML</td><td></td></tr><tr><td>Paraformaldehyde 4%</td><td>Sigma</td><td>P6148-500G&amp;nbsp;</td><td>Paraformaldehyde (4%) dissolved in PBS 1X&amp;nbsp;; aliquot by 600 ml&amp;nbsp;</td></tr><tr><td>PBS 1X</td><td>Thermo</td><td>14190094</td><td>DPBS, no calcium, no magnesium</td></tr><tr><td>0.1% (0.2 or 0.3%) PBS-T&amp;nbsp;</td><td></td><td></td><td>PBS 1X&amp;nbsp; containing 0.1% (0.2 % or 0.3%) Triton X-100</td></tr><tr><td>Triton X-100, for molecular biology,&amp;nbsp;</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GFP booster</td><td>Chromotek</td><td>gba488</td><td>1/200</td></tr><tr><td>Mouse anti-Mtor</td><td></td><td></td><td>1/40 gift from J. Johansen, Iowa State University</td></tr><tr><td>Rat anti-Nup62</td><td></td><td></td><td>1/200 gift from H. Ohkura (University of Edinburgh, UK</td></tr><tr><td>Guinea-pig anti-Ulp1</td><td></td><td></td><td>1/200 gift from C. P. Verrijzer, Erasmus University, Rotterdam</td></tr><tr><td>Rabbit anti-Raf2</td><td></td><td></td><td>1/200 gift from C. P. Verrijzer, Erasmus University, Rotterdam</td></tr><tr><td>DyLight conjugated series&amp;nbsp;</td><td>Thermo</td><td></td><td>1/500 Donkey anti-(guinea-pig, mouse,&amp;nbsp; rabbit or rat) as second antibodies</td></tr></tbody></table>

immunostaining of whole-mount drosophila testes for 3d confocal analysis of large spermatocytes

Drosophila testisleri kök hücre biyolojisi, nükleer mimari, meiosis ve sperm gelişimi dahil olmak üzere biyolojik süreçleri incelemek için güçlü bir model sistemidir. Bununla birlikte, tüm Drosophila testislerinin immünolabelingi genellikle antikor penetrasyonu nedeniyle lekelemenin önemli ölçüde homojen olmayanlığı ile ilişkilidir. Ezilmiş preparatlar, analizlerin 3D kalitesini azalttığı için sorunun sadece kısmen üstesinden gelmektedir. Burada, sıvı ortamda immünolabeling ile birlikte fiksasyon sırasında NP40 ve heptane kullanan tam montajlı bir protokol açıklıyoruz. Konfokal mikroskopiye uygun hacmi tekrarlanabilir ve güvenilir etiketleme ile birlikte korur. Konfokal bölümlerden spermatosit çekirdeğinin 3D rekonsesinin farklı örneklerini gösteriyoruz. İntra-ve inter-testes tekrarlanabilirliği, farklı genotiplerden tek hücreler arasındaki floresanların 3D nicelleştirilmesine ve karşılaştırılmasını sağlar. Bu protokolü ve testislerin en büyük hücreleri olan S4-S5 spermatositleri üzerindeki uygulamalarını göstermek için intranükleer MINT yapısının (Mad1 içeren Intra Nükleer Bölge) farklı bileşenlerinin yanı sıra nükleer gözenek kompleksi ile ilişkili iki bileşen kullandık.

Drosophila testislerinin yeterli lekelenmesine yönelik en büyük engel, ezilmiş preparatlar kullanılarak kısmen çözülmüş olan ancak 3D analizlerin (örneğin 3D temsili veya kolokalizasyon ölçüsü) kısıtlanması pahasına antikorların sınırlı penetrability'dir. Prosedür tek tip etiketlemeye izin veriyor ve testislerin tüm hücrelerinin hacmini koruyor. Burada yöntemin illüstrasyonunu S4-S5 spermatositlerinin 3D temsiline odakladık, çünkü en büyük hücrelerdir ve böylece ezilme deformasyonuna...

Watch this Scientific Journal Video about Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes at JoVE.com

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

Burada, konfokal mikroskopi için uygun ve aynı zamanda tekrarlanabilir ve güvenilir etiketlemeye izin vermek için Drosophila testislerinin tüm montaj hazırlığı ve immünostainingi için geliştirilmiş bir protokol sunuyoruz. Bu protokolü, büyük S4-S5 spermatositleri üzerindeki kolokalizasyon deneylerinin 3D gösterimi ve niceliği ile göstermektedir.

Büyük Spermatositlerin 3D Konfokal Analizi için Tüm Kazdağları Drosophila Testlerinin İmmünostaining

Drosophila testes are a powerful model system for studying biological processes including stem cell biology, nuclear architecture, meiosis and sperm ...

immunostaining-whole-mount-drosophila-testes-for-3d-confocal-analysis

Research

JoVE Journal

Biology

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

4.1K Görüntüleme.  Université de Paris, F 75006, Paris, France. Burada, konfokal mikroskopi için uygun ve aynı zamanda tekrarlanabilir ve güvenilir etiketlemeye izin vermek için Drosophila testislerinin tüm montaj hazırlığı ve immünostainingi için geliştirilmiş bir protokol sunuyoruz. Bu protokolü, büyük S4-S5 spermatositleri üzerindeki kolokalizasyon deneylerinin 3D gösterimi ve niceliği ile göstermektedir.

Video: Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

Upright Imaging of Drosophila Egg Chambers

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.

Upright Imaging of Drosophila Egg Chambers

The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

Dik Görüntüleme<em&gt; Drosophila</em&gt; Yumurta Odaları

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in ...

Gelişim Biyolojisi

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes&#8217; large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

The goal of this protocol is to analyze cell division in intact tissue by live and fixed cell microscopy using Drosophila meiotic spermatocytes. The protocol demonstrates how to isolate whole, intact testes from Drosophila larvae and early pupae, and how to process and mount them for microscopy.

Drosophila Larval ve Pupal Testislerinin Canlı, Sağlam Dokuda Hücre Bölünmesi Analizi İçin Hazırlanması

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...

Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells entering meiotic prophase I are eliminated during Fetal Oocyte Attrition (FOA) and the first week of postnatal life. Three major mechanisms regulate the number of oocytes that survive during development and establish the ovarian reserve in females entering puberty. In the first wave of oocyte loss, 30-50% of the oocytes are eliminated during early FOA, a phenomenon that is attributed to high Long interspersed nuclear element-1 (LINE-1) expression. The second wave of oocyte loss is the elimination of oocytes with meiotic defects by a meiotic quality checkpoint. The third wave of oocyte loss occurs perinatally during primordial follicle formation when some oocytes fail to form follicles. It remains unclear what regulates each of these three waves of oocyte loss and how they shape the ovarian reserve in either mice or humans.
Immunofluorescence and 3D visualization have opened a new avenue to image and analyze oocyte development in the context of the whole ovary rather than in less informative 2D sections. This article provides a comprehensive protocol for whole ovary immunostaining and optical clearing, yielding preparations for imaging using multiphoton microscopy and 3D modeling using commercially available software. It shows how this method can be used to show the dynamics of oocyte attrition during ovarian development in C57BL/6J mice and quantify oocyte loss during the three waves of oocyte elimination. This protocol can be applied to prenatal and early postnatal ovaries for oocyte visualization and quantification, as well as other quantitative approaches. Importantly, the protocol was strategically developed to accommodate high-throughput, reliable, and repeatable processing that can meet the needs in toxicology, clinical diagnostics, and genomic assays of ovarian function.

Here, we present an optimized protocol for imaging entire ovaries for quantitative and qualitative analyses using whole-mount immunostaining, multiphoton microscopy, and 3D visualization and analysis. This protocol accommodates high-throughput, reliable, and repeatable processing that is applicable for toxicology, clinical diagnostics, and genomic assays of ovarian function.

Farede Gelişmekte Olan Yumurtalık Rezervinin Kantitatif 3D Analizi için Tüm Yumurtalık İmmünofloresansı, Temizleme ve Multifoton Mikroskobu

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

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 ...

Biyoloji

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies 1-3. During pupation, which spans approximately 96 hours (at 25 &deg;C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4. Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

Drosophila pupa Karın İmmünohistokimya

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic ...

Imaging Centrosomes in Fly Testes

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell differentiation. Centrosomes are essential to determine the cell division axis during mitosis and to nucleate cilia during interphase. The identity of the proteins that mediate these dynamic changes remains only partially known, and the function of many of the proteins that have been implicated in these processes is still rudimentary. Recent work has shown that Drosophila spermatogenesis provides a powerful system to identify new proteins critical for centrosome function and formation as well as to gain insight into the particular function of known players in centrosome-related processes. Drosophila is an established genetic model organism where mutants in centrosomal genes can be readily obtained and easily analyzed. Furthermore, recent advances in the sensitivity and resolution of light microscopy and the development of robust genetically tagged centrosomal markers have transformed the ability to use Drosophila testes as a simple and accessible model system to study centrosomes. This paper describes the use of genetically-tagged centrosomal markers to perform genetic screens for new centrosomal mutants and to gain insight into the specific function of newly identified genes.

Imaging of centrosomal proteins during Drosophila spermatogenesis is a powerful method to identify new proteins critical for centrosome biology as well as to elucidate the particular function of known players in this process.

Fly Testislerin görüntüleme centrosomes

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

Spermatogenez sitolojik analizi: Canlı ve Sabit Hazırlıklar<em&gt; Drosophila</em&gt; Testisler

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

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 ...

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin&#8212;the histone-to-protamine switch.
Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.
The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.

Diseksiyon, Görüntüleme ve Farmakolojik Tedavi: Drosophila Pupa Testis ve Bekar Erkek germ-line Kistler ex vivo Kültür

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes ...

Super-Resolution Live Cell Imaging of Subcellular Structures

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to.
Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres.
This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

Hücre Altı Yapıların Süper Çözünürlüklü Canlı Hücre Görüntülemesi

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...

Büyük Spermatositlerin 3D Konfokal Analizi için Tüm Kazdağları Drosophila Testlerinin İmmünostaining

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