Name: immunostaining of whole-mount drosophila testes for 3d confocal analysis of large spermatocytes
Uploaded: 2020-08-27T14:00:00.000+00:00
Duration: 8 min 37 s
Description: Watch this Scientific Journal Video about Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes at JoVE.com

Wir danken P. Verrijzer, J. Johansen H., Okhura für Antikörper und A. Debec, den Lagerzentren Bloomington und Vienna für Fliegen. Danke an C. Jackson für Anregungen und anregende Diskussionen.

1. Drosophila Hodensammlung
HINWEIS: Junge Männchen (0-15 h nach der Eklosion) sind notwendig, um diploide Keimzellen (dh Spermatogonie und Spermatozyten) zu untersuchen.
<ol><li>Wählen Sie junge Fliegen so weit wie möglich aus, um Störungen durch die Entwicklung von Spermatiden zu minimieren.</li>
	<li>Anästhesieren Sie Fliegen durch kurze Exposition gegenüber Kohlendioxid und Übertragen auf ein Fliegenpad14,15.</li>
	<li>Unter einem Seziermikroskop (10-fache Vergrößerung) den Kopf mit einer Zette entfernen.</li>
	<li>5 bis 10 enthauptete Fliegen auf 100 μL Ringer-Puffer (183 mM KCI, 47 mM NaCl, 10 mM Tris-HCI, 1 mM EDTA, vollständiger Proteininhibitor, pH 6,8) auf einen silikonbeschichteten Glasträger auf schwarzem Hintergrundträger übertragen.</li>
	<li>Verwenden Sie mit einem Seziermikroskop bei einer 40-fachen Vergrößerung ein Paar Einer Zange Nummer 5, um eine Fliege zwischen Thorax und Bauch zu halten, und ziehen Sie mit einer anderen Zange den Bauch vom Thorax weg. Isolieren Sie schnell die Hoden und das angrenzende Gewebe aus dem Fliegenkadaver15 und legen Sie einen neuen Tropfen Ringers Puffer auf denselben Objektträger.</li>
	<li>Sobald alle Hoden fertig sind, reinigen Sie die Hoden von den verbleibenden Geweben (Akzessodrüse und äußere Genitalien).</li>
	<li>Entfernen Sie überschüssigen Puffer aus dem Tropfen, der die sezierten Hoden enthält. Verwenden Sie in der Regel eine p200-Pipette mit einer 10-μL-Spitze, die auf einer 200-μL-Spitze angebracht ist. Dies ermöglicht die Entfernung des Flüssigkeitsüberschusses durch eine kleine Öffnung. Legen Sie einen Tropfen 4% Paraformaldehyd-Fixiermittel auf den Tropfen, der die sezierten Hoden enthält, und geben Sie die Hoden dann schnell in ein Röhrchen mit frischer Fixierlösung.</li>
</ol>2. Paraformaldehyd-Fixierung
<ol><li>Bereiten Sie 2 ml Fixierlösung kurz vor dem Gebrauch vor: 600 μL 4% Paraformaldehyd verdünnt in phosphatgepufferter Kochsalzlösung (PBS) mit 30 μL NP40 20% plus 1200 μL Heptan in einem 2 mL silikonisierten Mikrozentrifugenröhrchen. Verwenden Sie 4% Paraformaldehyd in PBS, das in 600 μL Aliquoten hergestellt und bei -20 °C gelagert wird. Achtung: Heptan und Paraformaldehyd sind giftig und sollten in einem Abzug gehandhabt werden. Entsorgen Sie diese gemäß den Vorschriften des Gastinstituts.</li>
	<li>Übertragen Sie die Hoden mit einer p200-Mikropipette mit einer breiten Öffnungsspitze auf die PFA/NP40/Heptan-Lösung. Schütteln Sie den Schlauch 30 s lang von Hand auf und ab. Setzen Sie die Fixierung der Hoden auf einem Rotationsmischer bei Raumtemperatur für 30 minuten fort.</li>
	<li>Stoppen Sie den Rotationsmischer und legen Sie das Rohr in ein Rohrgestell, damit die Phasentrennung erfolgt und sich die Hoden am Boden des Rohres absetzen können.</li>
	<li>Entfernen Sie das gesamte Heptan und die Fixierlösung und spülen Sie die Hoden schnell dreimal mit 1x PBS aus.</li>
	<li>In ein sauberes 200 μL Einzelröhrchen überführen. Führen Sie alle Inkubationen in diesen kleinen Röhrchen durch, um die Menge der verwendeten Antikörper zu minimieren.</li>
	<li>Entfernen Sie überschüssigen Puffer mit einer 200 μL-Mikropipette, an der sowohl eine P200- als auch eine P10-Spitze angebracht sind. Achten Sie darauf, keine Hoden anzusaugen.</li>
	<li>Halten Sie die Hoden in 1x PBS auf Eis, bis alle Proben fertig sind.</li>
</ol>3. Antikörperfärbung in Lösung
<ol><li>Verwerfen Sie das PBS und lassen Sie die Hoden in 0,3% PBS-T für 30 minuten bei Raumtemperatur, um die Zellmembranen zu permeabilisieren.</li>
	<li>Im selben Puffer plus 5% normales Ziegenserum (NGS) für 1 h vorinkubieren.</li>
	<li>200 μL des primären Antikörpers verdünnt in 0,3% PBS-T plus 5% NGS ( Materialtabelle )werden hinzugefügt.</li>
	<li>Inkubieren Sie den primären Antikörper über Nacht bei 4 °C (oder Raumtemperatur) mit sanftem Rühren auf einer Wippe.</li>
	<li>3 mal in 0,3% PBS-T für 15 min auf einer Wippe bei Raumtemperatur waschen. Lassen Sie die Hoden sich durch die Schwerkraft auf den Boden der Röhre absetzen.</li>
	<li>Fügen Sie 200 μL sekundären Antikörper hinzu, der 1:500 verdünnt ist, aus der dyLight-konjugierten Serie.</li>
	<li>Inkubieren Sie den sekundären Antikörper in einer dunklen Kammer für 4 h bei Raumtemperatur.</li>
	<li>In 0,3% PBS-T für 15 min auf einer Wippe bei Raumtemperatur waschen. Dann mit 0,2% PBS-T und 0,1% PBS-T wiederholen, jedes Mal für 30 minuten auf einer Wippe bei Raumtemperatur. Waschen Sie das Reinigungsmittel abschließend in 1x PBS für 30 Minuten, um das Reinigungsmittel zu entfernen.</li>
	<li>180 μL DAPI-Lösung bei 1 μg/ml in 1x PBS für 15 min hinzufügen. Die Stammlösung beträgt 10 mg/ ml inH2O. Vermeiden Sie die Herstellung von Stammlösung in PBS, die das DAPI-Färbesignal auf diffusem Chromatin in größeren Spermatozyten reduzieren kann.</li>
	<li>3x für je 5 min waschen.</li>
	<li>Verwenden Sie einen hydrophoben Barrierestift, um einen Kreis auf einem sauberen Dia zu zeichnen.</li>
	<li>Saugen Sie hoden an und legen Sie sie auf den Schlitten. Das Vorspülen der weiten Blendenspitze mit 0,1% PBS-T verhindert, dass die Hoden an der Spitze haften. Entfernen Sie überschüssiges PBS.</li>
	<li>Fügen Sie einen Tropfen (ca. 100 μL) Citifluor AF1 hinzu.</li>
	<li>Legen Sie vorsichtig einen Glasdeckel über das Gewebe und achten Sie darauf, dass Luftblasen nicht einfangen.</li>
	<li>Versiegeln Sie den Abdeckverschluss mit klarem Nagellack auf dem Schlitten.</li>
	<li>Beobachten Sie Objektträger auf einem Mikroskop.</li>
</ol>4. Kolokalisationsanalysen
<ol><li>Erfassen Sie konfokale Bilder. Wir verwendeten ein Zeiss LSM 710 Konfokalmikroskop (63x Plan Apochromatic Öl) mit einer z-Auflösung von 0,5 oder 1 μm.</li>
	<li>Wählen Sie verschiedene Zellen aus unabhängigen Hoden aus.</li>
	<li>Importieren Sie Bilder in Verarbeitungssoftware (z. B. Imaris).</li>
	<li>Definieren Sie den Kern durch manuelle Segmentierung, um die benachbarten Kerne auszuschließen. Verwenden Sie die Coloc-Software; Es sammelt die Pearson-Korrelationskoeffizienten (PCCs) innerhalb des gesamten Volumens zwischen 2 Fluorophoren.</li>
</ol>

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Dieses Protokoll bietet eine Methode zur erfolgreichen Immunmarkierung von drosophila-Hoden bei Erwachsenen. Wie in anderen Verfahren berichtet, müssen junge Männer verwendet werden (wir haben sogar das Alter auf weniger als 20 Stunden nach der Eklosion verkürzt), um das Vorhandensein von sich entwickelnden Spermien zu minimieren, die die Spermatozyten behindern könnten. Die Dissektion sollte schnell sein und die Antikörperverdünnung muss angepasst werden, um das Signal-Rausch-Verhältnis21<...

Drosophila-Hoden sind ein wertvolles Modell für das Studium der nuklearen Architektur. In mitotischen Spermatogonienzellen ist das Kernvolumen weitgehend mit Chromatin besetzt. Diese Zellen durchlaufen vier Teilungsrunden und produzieren eine Zyste von 16 primären Spermatozyten. In den nächsten Tagen durchlaufen die Spermatozyten 6 Entwicklungsstadien (S1-S6), wobei das Volumen stark zunimmt, ungefähr 20-fach1,2. Sie verändern auch ihre Kernmorphologie. Der Umriss des Kerns, der durch das Lamin Dm0 offenbart wird, wird unregelmäßig und zeigt tiefe Invaginationen1,3. Damit einher gehen umfangreiche Genexpressionsveränderungen und Modifikationen in der Chromatinorganisation1,4,5,6,7. Die Interphasenchromosomen sind im Stadium S4-S5 gut definiert und bilden drei verschiedene Massen, die den 2 wichtigsten bivalenten Autosomen und dem X-Y-Paar entsprechen, das an den Nukleolus angedockt ist.
Mad1 wurde ursprünglich als Schlüsselkomponente des mitotischen Checkpoints isoliert (überprüft in8). In der Interphase wird es als primär an der Kernhülle, aber auch im Nukleoplasmus9,10,11lokalisiert beschrieben. Bei Drosophila hoden berichteten wir, dass Mad1 im Nukleoplasma prominent ist, eng mit den beiden großen autosomalen Chromatinmassen und in geringerem Maße mit dem XY-Chromatin verbunden ist. Wir haben diese Chromatin-assoziierte Struktur als MINT (Mad1-containing IntraNuclear Territory)12definiert. Vier weitere Proteine waren mit MINTs in Spermatozyten assoziiert: das Nukleoporin Mtor/Tpr, das SUMO peptidase Ulp1, das mitotische Checkpointprotein Mad2 und eine Untereinheit eines Polycomb-ähnlichen Komplexes Raf212.
Um die Beschreibung von MINTs zu vervollständigen, mussten wir Antikörper verwenden und wurden mit den technischen Problemen konfrontiert, die im Allgemeinen bei der Immunfärbung im Hoden auftreten: eine schlechte Permeabilität, die zu einer signifikanten Ungleichmäßigkeit der Färbung in der Tiefe des Hodens, insbesondere in den großen Spermatozyten, führt. Szerschmierte Präparate erhöhten den Antikörperzugang, führten jedoch zu komprimierten Bildern, schränkten die 3D-Analysen ein und verhinderten die Messung der quantitativen Fluoreszenz, einschließlich der Kolokalisierung. Das Problem der Antikörperpenetration könnte auch durch Permeabilisierungsmittel wie 0,3% Na-Desoxycholat13angegangen werden, aber dieses Verfahren führte in unseren Händen nicht zu reproduzierbaren Ergebnissen. Da wir viele Co-Labeling-Experimente durchgeführt haben, haben wir versucht, das Permeabilisierungsprotokoll zu verbessern. Die Kombination von 1% NP40 plus Heptan führte zu mehr Reproduzierbarkeit, insbesondere bei der Untersuchung der großen Spermatozyten.
Dieses Protokoll führt die Fixierung und Immunfärbung von Hoden in Suspension durch, verbessert die Probenqualität sowie verbessert die Reproduzierbarkeit und macht sie für die konfokale Mikroskopie geeignet. Wir haben das Protokoll verwendet, um die räumlichen Beziehungen bestimmter Kernhüllenproteine mit den nukleoplasmatischen Strukturen großer Spermatozyten besser zu definieren. Diese Methode wird es ermöglichen, die Veränderungen, die die Spermatozytenentwicklung begleiten, besser zu überprüfen, zum Beispiel die dynamischen strukturellen Veränderungen des Kerns einschließlich Chromatin, Nukleoplasma und der Kernporen.

<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-Hoden sind ein leistungsfähiges Modellsystem zur Untersuchung biologischer Prozesse, einschließlich Stammzellbiologie, Kernarchitektur, Meiose und Spermienentwicklung. Die Immunmarkierung des gesamten Drosophila-Hodens ist jedoch oft mit einer signifikanten Ungleichmäßigkeit der Färbung aufgrund der Antikörperpenetration verbunden. Gequetschte Präparate überwinden das Problem nur teilweise, da sie die 3D-Qualität der Analysen verringern. Hierin beschreiben wir ein Whole-Mount-Protokoll mit NP40 und Heptan während der Fixierung zusammen mit der Immunmarkierung in flüssigen Medien. Es bewahrt das für die konfokale Mikroskopie geeignete Volumen zusammen mit einer reproduzierbaren und zuverlässigen Markierung. Wir zeigen verschiedene Beispiele für die 3D-Rekonstitution von Spermatozytenkernen aus konfokalen Abschnitten. Die Reproduzierbarkeit von Intra- und Inter-Hoden ermöglicht die 3D-Quantifizierung und den Vergleich der Fluoreszenz zwischen einzelnen Zellen verschiedener Genotypen. Wir verwendeten verschiedene Komponenten der intranukleären MINT-Struktur (Mad1-containing Intra Nuclear Territory) sowie zwei Komponenten, die mit dem Kernporenkomplex verbunden sind, um dieses Protokoll und seine Anwendungen auf den größten Zellen des Hodens, den S4-S5-Spermatozyten, zu veranschaulichen.

Das Haupthindernis für eine angemessene Färbung der Drosophila-Hoden ist die begrenzte Penetrabilität von Antikörpern, die teilweise durch die Verwendung von gequetschten Präparaten gelöst wurde, aber auf Kosten einer Einschränkung der 3D-Analysen (z. B. 3D-Darstellung oder Maß der Kolokalisierung). Das Verfahren ermöglicht eine einheitliche Kennzeichnung und bewahrt das Volumen aller Zellen des Hodens. Hier haben wir die Darstellung der Methode auf die 3D-Darstellung von S4-S5-Spermatozyten konzentriert, da die...

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

Wir präsentieren hier ein verbessertes Protokoll für die Ganzkörperpräparation und Immunfärbung von Drosophila-Hoden, das für die konfokale Mikroskopie geeignet ist und auch eine reproduzierbare und zuverlässige Markierung ermöglicht. Wir veranschaulichen dieses Protokoll durch 3D-Darstellung und Quantifizierung von Kolokalisierungsexperimenten an den großen S4-S5-Spermatozyten.

Immunfärbung von Drosophila-Hoden für die konfokale 3D-Analyse großer Spermatozyten

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 Aufrufe.  Université de Paris, F 75006, Paris, France. Wir präsentieren hier ein verbessertes Protokoll für die Ganzkörperpräparation und Immunfärbung von Drosophila-Hoden, das für die konfokale Mikroskopie geeignet ist und auch eine reproduzierbare und zuverlässige Markierung ermöglicht. Wir veranschaulichen dieses Protokoll durch 3D-Darstellung und Quantifizierung von Kolokalisierungsexperimenten an den großen S4-S5-Spermatozyten.

Serie: 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.

Upright-Imaging von<em&gt; Drosophila</em&gt; Egg Chambers

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

Forschung

Entwicklungsbiologie

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.

Vorbereitung von Drosophila Larval und Pupal Hoden für die Analyse der Zellteilung in Leben, intaktes Gewebe

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.

Immunfluoreszenz, Clearing und Multiphotonenmikroskopie des gesamten Eierstocks zur quantitativen 3D-Analyse der sich entwickelnden Ovarialreserve in der Maus

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.

Dissection und Färbung von Drosophila Larven Ovarien

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

Biologie

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 Puppenstadium Abdomen Immunhistochemie

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.

Imaging Zentrosomen in Fly Hoden

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;

Die zytologische Analyse der Spermatogenese: Live-und Fest Zubereitungen aus<em&gt; Drosophila</em&gt; Hoden

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.

Beschallung-erleichtert Immunfluoreszenzfärbungen der Late-Stage embryonalen und Larven<em&gt; Drosophila</em&gt; Gewebe<em&gt; In-situ</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.

Ex-vivo-Kultur von Drosophila Pupal Hoden und Single männlichen Keim Online-Zysten: Dissection, Imaging und pharmakologische Behandlung

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.

Hochauflösende Live-Cell-Bildgebung subzellulärer Strukturen

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

Immunfärbung von Drosophila-Hoden für die konfokale 3D-Analyse großer Spermatozyten

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