Ringraziamo P. Verrijzer, J. Johansen H., Okhura per gli anticorpi e A. Debec, i centri di borsa bloomington e vienna per le mosche. Grazie a C. Jackson per suggerimenti e discussioni stimolanti.

1. Raccolta testes Drosophila
NOTA: I giovani maschi (0-15 ore dopo l'eclosion) sono necessari per esaminare le cellule germinali diploidi (ad esempio spermatogonia e spermatociti).
<ol><li>Seleziona le mosche giovani il più possibile per ridurre al minimo le interferenze derivanti dallo sviluppo di spermatidi.</li>
	<li>Anestetizza le mosche con una breve esposizione all'anidride carbonica e si trasferisce su un fly pad14,15.</li>
	<li>Al microscopio sezionato (ingrandimento 10x), utilizzare le flessione per rimuovere la testa.</li>
	<li>Trasferire da 5 a 10 mosche decapitate a 100 μL del tampone di Ringer (183 mM KCI, 47 mM NaCl, 10 mM Tris-HCI, 1 mM EDTA, inibitore proteico completo, pH 6,8) su uno scivolo di vetro rivestito di silicone su un supporto di sfondo nero.</li>
	<li>Con il microscopio sezionato con un ingrandimento di 40x, utilizzare un paio di forcep numero 5 per tenere una mosca tra il torace e l'addome e, con altre forcep, allontanare l'addome dal torace. Isolare rapidamente i tester e i tessuti adiacenti dalla carcassadi mosca 15 e posizionare in una nuova goccia del buffer di Ringer sullo stesso scivolo.</li>
	<li>Una volta che tutti i teste sono pronti, pulire i teste dai tessuti rimanenti (ghiandola accessoria e genitali esterni).</li>
	<li>Rimuovere il buffer in eccesso dalla goccia contenente i teste sezionati. In genere, utilizzare una pipetta p200 con una punta da 10 μL montata sopra una punta da 200 μL. Ciò consente la rimozione dell'eccesso di liquido attraverso una piccola apertura. Posizionare una goccia del 4% di paraformaldeide fissativa sopra la goccia contenente i tester sezionati, quindi trasferire rapidamente i tester su un tubo contenente soluzione fissante fresca.</li>
</ol>2. Fissazione della paraformaldeide
<ol><li>Preparare 2 ml di soluzione fissativa poco prima dell'uso: 600 μL di paraformaldeide al 4% diluita in salina tamponata di fosfato (PBS) con 30 μL di NP40 20% più 1200 μL di eptano in un tubo di microcentrifugo siliconato da 2 ml. Utilizzare il 4% di paraformaldeide in PBS preparata in aliquote 600 μL e conservata a -20°C. Attenzione: Eptano e paraformaldeide sono tossici e devono essere maneggiati in una cappa aspirante. Smaltirli secondo il regolamento dell'istituto ospitante.</li>
	<li>Trasferire i tester utilizzando una micropipetta p200 con un'ampia punta di apertura alla soluzione PFA/NP40/eptano. Agitare il tubo su e giù per 30 s a mano. Continuare a fissare i tester su un miscelatore rotativo a temperatura ambiente per 30 minuti.</li>
	<li>Arrestare il miscelatore rotante e posizionare il tubo in un rack per tubi per consentire la separazione di fase e per i tester di depositarsi sul fondo del tubo.</li>
	<li>Rimuovere tutto l'eptano e la soluzione fissante e risciacquare rapidamente i tester tre volte con 1x PBS.</li>
	<li>Trasferire in un singolo tubo pulito da 200 μL. Eseguire tutte le incubazioni in questi piccoli tubi per ridurre al minimo le quantità di anticorpi impiegati.</li>
	<li>Rimuovere il buffer in eccesso utilizzando una micropipetta da 200 μL con una punta P200 e una punta P10 attaccata. Fare attenzione a non aspirare teste.</li>
	<li>Conservare i test in 1x PBS sul ghiaccio fino a quando tutti i campioni non sono pronti.</li>
</ol>3. Colorazione anticorpale in soluzione
<ol><li>Scartare il PBS e lasciare i tester nello 0,3% di PBS-T per 30 minuti a temperatura ambiente per permeabilizzare le membrane cellulari.</li>
	<li>Pre-incubare nello stesso tampone più il 5% di siero di capra normale (NGS) per 1 h.</li>
	<li>Aggiungere 200 μL dell'anticorpo primario diluito nello 0,3% pbs-t più 5% NGS(Tabella dei materiali).</li>
	<li>Incubare l'anticorpo primario durante la notte a 4 °C (o temperatura ambiente) con delicata agitazione su un rocker.</li>
	<li>Lavare 3 volte in 0,3% PBS-T per 15 minuti su un rocker a temperatura ambiente. Lasciare che i tester si sistemino sul fondo del tubo per gravità.</li>
	<li>Aggiungere 200 μL di anticorpo secondario diluito 1:500 dalla serie coniugata DyLight.</li>
	<li>Incubare l'anticorpo secondario in una camera scura per 4 ore a temperatura ambiente.</li>
	<li>Lavare in 0,3% PBS-T per 15 minuti su un rocker a temperatura ambiente. Quindi ripetere con 0,2% PBS-T e 0,1% PBS-T, ogni volta per 30 minuti su un rocker a temperatura ambiente. Fare un lavaggio finale in 1x PBS per 30 minuti per rimuovere il detergente.</li>
	<li>Aggiungere 180 μL di soluzione DAPI a 1 μg/mL in 1x PBS per 15 min. La soluzione stock è di 10 mg/mL in H2O. Evitare di fare soluzione stock in PBS, che può ridurre il segnale di colorazione DAPI sulla cromatina diffusa negli spermatociti più grandi.</li>
	<li>Lavare 3 volte per 5 minuti ciascuno.</li>
	<li>Utilizzare una penna barriera idrofobica per disegnare un cerchio su uno scivolo pulito.</li>
	<li>Aspirare i teste e metterli sulla diapositiva. Il preri risciacquo della punta di apertura ampia con PBS-T dello 0,1% impedisce ai tester di attaccarsi alla punta. Rimuovere il PBS in eccesso.</li>
	<li>Aggiungere una goccia (circa 100 μL) di Citifluor AF1.</li>
	<li>Posizionare delicatamente una coverlip di vetro sul tessuto, facendo attenzione a evitare di intrappolare bolle d'aria.</li>
	<li>Sigillare il coverslip allo scivolo utilizzando smalto chiaro.</li>
	<li>Osservare le diapositive al microscopio.</li>
</ol>4. Analisi di colocalizzazione
<ol><li>Acquisire immagini confocali. Abbiamo usato un microscopio confocale Zeiss LSM 710 (olio apocromatico a piano 63x) con una risoluzione z di 0,5 o 1 μm.</li>
	<li>Selezionare celle diverse da tester indipendenti.</li>
	<li>Importare immagini nel software di elaborazione (ad esempio, Imaris).</li>
	<li>Definire il nucleo mediante segmentazione manuale per escludere i nuclei vicini. Utilizzare il software Coloc; raccoglie i coefficienti di correlazione di Pearson (PCC) all'interno dell'intero volume tra 2 fluorofori.</li>
</ol>

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Gli autori non hanno nulla da rivelare.

Questo protocollo fornisce un metodo per l'immunoetichettatura di tester di Drosophila adulti a montaggio intero. Come riportato in altre procedure devono essere utilizzati giovani maschi (abbiamo anche ridotto l'età a meno di 20 ore dopo l'eclosion) per ridurre al minimo la presenza di spermatozoi in via di sviluppo, che rischia di ostruire gli spermatociti. La dissezione deve essere rapida e la diluizione degli anticorpi deve essere regolata per ottimizzare il rapporto segnale-rumore21,</s...

I test di Drosophila sono un modello prezioso per lo studio dell'architettura nucleare. Nelle cellule spermatogoniche mitotiche, il volume nucleare è in gran parte occupato dalla cromatina. Queste cellule subiscono quattro cicli di divisione, producendo una cisti di 16 spermatociti primari. Nei giorni successivi, gli spermatociti passano attraverso 6 stadi di sviluppo (S1-S6), aumentando notevolmente di volume, approssimativamente 20 volte1,2. Cambiano anche la loro morfologia nucleare. Il contorno del nucleo, rivelato dal lamina Dm0, diventa irregolare e mostra profonde invaginazioni1,3. Questo è accompagnato da ampi cambiamenti di espressione genica e modifiche nell'organizzazione della cromatina1,4,5,6,7. I cromosomi interfase sono ben definiti nello stadio S4-S5, formando tre masse distinte corrispondenti ai 2 principali autosomi bivalenti e alla coppia X-Y agganciata al nucleolo.
Mad1 è stato originariamente isolato come componente chiave del checkpoint mitotico (esaminato in8). Nell'interfase, è descritto come situato principalmente all'involucro nucleare ma anche nel nucleoplasma9,10,11. In Drosophila testis, abbiamo riferito che Mad1 è prominente nel nucleoplasma, intimamente associato alle due principali masse di cromatina autosomica e in misura minore alla cromatina XY. Abbiamo definito questa struttura associata alla cromatina come MINT (Mad1-containing IntraNuclear Territory)12. Altre quattro proteine erano associate ai TT negli spermatociti: la nucleoporina Mtor/Tpr, la peptidasi Ulp1 suMO, la proteina del checkpoint mitotico Mad2 e una subunità di un complesso simile a Policomb Raf212.
Per completare la descrizione dei MINT, dovevamo usare gli anticorpi e ci siamo trovati di fronte ai problemi tecnici generalmente incontrati con l'immunostaining nel testicolo: una scarsa permeabilità con conseguente significativa non uniformità di colorazione nella profondità del testicolo, in particolare nei grandi spermatociti. Ipreparati s annullati aumentavano l'accesso agli anticorpi ma portavano a immagini compresse, limitando le analisi 3D e impedendo la misurazione della fluorescenza quantitativa, compresa la colocalizzazione. Il problema della penetrazione degli anticorpi potrebbe anche essere affrontato da agenti permeabilizzanti come lo 0,3% di Na deossicholato13, ma questa procedura non ha prodotto nelle nostre mani risultati riproducibili. Poiché abbiamo eseguito molti esperimenti di co-etichettatura, abbiamo cercato di migliorare il protocollo di permeabilizzazione. La combinazione di 1% NP40 più eptano ha portato a una maggiore riproducibilità, in particolare nello studio dei grandi spermatociti.
Questo protocollo esegue la fissazione e l'immunosocienza dei test in sospensione, migliorando la qualità del campione e migliorando la riproducibilità e rendendolo adatto per la microscopia confocale. Abbiamo usato il protocollo per definire meglio le relazioni spaziali di alcune proteine dell'involucro nucleare con le strutture nucleoplasmatiche dei grandi spermatociti. Questo metodo consentirà di esaminare meglio i cambiamenti che accompagnano lo sviluppo degli spermatociti, ad esempio i cambiamenti strutturali dinamici del nucleo, tra cui cromatina, nucleoplasma e pori nucleari.

<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

I test di Drosophila sono un potente sistema modello per lo studio dei processi biologici tra cui la biologia delle cellule staminali, l'architettura nucleare, la meiosi e lo sviluppo dello sperma. Tuttavia, l'immunoetichettatura dell'intero testicolo drosophila è spesso associata a una significativa non uniformità di colorazione dovuta alla penetrazione degli anticorpi. I preparati schiacciati superano solo parzialmente il problema poiché diminuisce la qualità 3D delle analisi. Qui, descriviamo un protocollo a montaggio intero usando NP40 ed eptano durante la fissazione insieme all'immunoetichettatura nei mezzi liquidi. Preserva il volume adatto alla microscopia confocale insieme a etichette riproducibili e affidabili. Mostriamo diversi esempi di ricostituzione 3D dei nuclei di spermatociti da sezioni confocali. La riproducibilità intra- e inter-testes consente la quantificazione 3D e il confronto della fluorescenza tra singole cellule di diversi genotipi. Abbiamo utilizzato diversi componenti della struttura MINT intranucleare (Mad1-containing Intra Nuclear Territory) e due componenti associati al complesso dei pori nucleari per illustrare questo protocollo e le sue applicazioni sulle più grandi cellule del testicolo, gli spermatociti S4-S5.

Il principale ostacolo all'ottenimento di un'adeguata colorazione dei test di Drosophila è la limitata penetrabilità degli anticorpi, che è stata parzialmente risolta utilizzando preparati schiacciati ma a costo di indurre una restrizione delle analisi 3D (ad esempio rappresentazione 3D o misura della colocalizzazione). La procedura consente un'etichettatura uniforme e preserva il volume di tutte le cellule del testicolo. Qui abbiamo focalizzato l'illustrazione del metodo sulla rappresentazione 3D degli spermatociti S...

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

Presentiamo qui un protocollo migliorato per la preparazione a montaggio intero e l'immunostaining dei tester Drosophila, adatto per la microscopia confocale e che consente anche un'etichettatura riproducibile e affidabile. Illustriamo questo protocollo attraverso la rappresentazione 3D e la quantificazione degli esperimenti di colocalizzazione sui grandi spermatociti S4-S5.

Immunostaining dei test di Drosophila a montaggio intero per l'analisi confocale 3D di grandi spermatociti

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 Views.  Université de Paris, F 75006, Paris, France. Presentiamo qui un protocollo migliorato per la preparazione a montaggio intero e l'immunostaining dei tester Drosophila, adatto per la microscopia confocale e che consente anche un'etichettatura riproducibile e affidabile. Illustriamo questo protocollo attraverso la rappresentazione 3D e la quantificazione degli esperimenti di colocalizzazione sui grandi spermatociti S4-S5.

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.

Imaging Upright di<em&gt; Drosophila</em&gt; Chambers Egg

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

Ricerca

Biologia dello sviluppo

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.

Preparazione dei test di Drosophila Larval e Pupal per l'analisi della divisione cellulare in live, tessuto intatto

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

Transillumination-Assisted Dissection of Specific Stages of the Mouse Seminiferous Epithelial Cycle for Downstream Immunostaining Analyses

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. Differentiation of germ cells takes place in the cytoplasmic pockets of somatic Sertoli cells that host 4 to 5 generations of germ cells simultaneously and coordinate and synchronize their development. Therefore, the composition of germ cell types within a cross-section is constant, and these cell associations are also known as stages (I&#8211;XII) of the seminiferous epithelial cycle. Importantly, stages can also be identified from intact seminiferous tubules based on their differential light absorption/scatter characteristics revealed by transillumination, and the fact that the stages follow each other along the tubule in a numerical order. This article describes a transillumination-assisted microdissection method for the isolation of seminiferous tubule segments representing specific stages of mouse seminiferous epithelial cycle. The light absorption pattern of seminiferous tubules is first inspected under a dissection microscope, and then tubule segments representing specific stages are cut and used for downstream applications. Here we describe immunostaining protocols for stage-specific squash preparations and for intact tubule segments. This method allows a researcher to focus on biological events taking place at specific phases of spermatogenesis, thus providing a unique tool for developmental, toxicological, and cytological studies of spermatogenesis and underlying molecular mechanisms.

This protocol describes transillumination-assisted microdissection of segments of adult mouse seminiferous tubules representing specific stages of seminiferous epithelial cycle, and cell types therein, and subsequent immunostaining of squash preparations and intact tubule segments.

Dissezione assistita da transilluminazione di stadi specifici del ciclo epiteliale seminifero del topo per analisi immunosottenifere a valle

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. ...

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.

Immunofluorescenza dell'ovaio intero, clearing e microscopia multifotonica per l'analisi quantitativa 3D della riserva ovarica in via di sviluppo nel topo

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.

Dissezione e colorazione di Drosophila Larvali ovaie

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

Biologia

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.

I centrosomi Imaging in Fly Testicoli

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;

L&#39;analisi citologica della spermatogenesi: Preparati Live e fisse di<em&gt; Drosophila</em&gt; Testicoli

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.

Sonicazione-facilitato immunofluorescenza colorazione di ritardo-fase embrionale e larvale<em&gt; Drosophila</em&gt; Tessuti<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 Cultura della Drosophila pupa testicolo e singolo maschio cisti germinale: Dissezione, Imaging, e trattamento farmacologico

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.

Imaging di cellule vive super-risoluzione di strutture subcellulari

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

Immunostaining dei test di Drosophila a montaggio intero per l'analisi confocale 3D di grandi spermatociti

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