Agradecemos a P. Verrijzer, J. Johansen H., Okhura por los anticuerpos y A. Debec, los centros de stock de Bloomington y Viena para las moscas. Gracias a C. Jackson por sus sugerencias y discusiones estimulantes.

1. Colección de testículos de Drosophila
NOTA: Los varones jovenes (0-15 h poste-eclosion) son necesarios para examinar las células de germen diploides (es decir, espermatogonia y spermatocytes).
<ol><li>Seleccione las moscas jóvenes tanto como sea posible para minimizar la interferencia del desarrollo de espezátidas.</li>
	<li>Anestesiar a las moscas por breve exposición al dióxido de carbono y transferir a una almohadilla de mosca14,15.</li>
	<li>Bajo un microscopio de disección (aumento de 10x), use fórceps para extraer la cabeza.</li>
	<li>Transfiera de 5 a 10 moscas decapitadas a 100 μL del tampón de Ringer (183 mM KCI, 47 mM NaCl, 10 mM Tris-HCI, 1 mM EDTA, inhibidor completo de proteínas, pH 6.8) en un portaobjetos de vidrio recubierto de silicona sobre un soporte de fondo negro.</li>
	<li>Con el microscopio de disección con un aumento de 40x, use un par de pórceps número 5 para sostener una mosca entre el tórax y el abdomen y, con otros tórceps, aleje el abdomen del tórax. Aísle rápidamente los testículos y los tejidos adyacentes de la canal de la mosca15 y colóquelos en una nueva gota del tampón de Ringer en el mismo portaobjetos.</li>
	<li>Una vez que todos los testículos estén listos, limpie los testículos de los tejidos restantes (glándula accesoria y genitales externos).</li>
	<li>Elimine el exceso de búfer de la gota que contiene los testículos disecados. Por lo general, utilice una pipeta p200 con una punta de 10 μL instalada encima de una punta de 200 μL. Esto permite la eliminación del exceso de líquido a través de una pequeña abertura. Coloque una gota de fijador de paraformadehído al 4% en la parte superior de la gota que contiene los testículos disecados, y luego transfiera rápidamente los testículos a un tubo que contenga una solución fijadora fresca.</li>
</ol>2. Fijación de paraformadehído
<ol><li>Preparar 2 mL de solución fijadora justo antes de su uso: 600 μL de paraformadehído al 4% diluido en solución salina tamponada con fosfato (PBS) con 30 μL de NP40 al 20% más 1200 μL de heptano en un tubo de microcentrífuga siliconado de 2 mL. Utilizar paraformadehído al 4% en PBS preparado en alícuotas de 600 μL y almacenado a -20°C. Precaución: El heptano y el paraformaldehído son tóxicos y deben manipularse en una campana extractora de humos. Disponer de ellos de acuerdo con las regulaciones del instituto anfitrión.</li>
	<li>Transfiera los testículos usando una micropipeta p200 con una punta de apertura amplia a la solución PFA/NP40/heptano. Agite el tubo hacia arriba y hacia abajo durante 30 s a mano. Continuar fijando los testículos en una batidora rotativa a temperatura ambiente durante 30 min.</li>
	<li>Detenga la mezcladora rotativa y coloque el tubo en un estante de tubos para permitir la separación de fases y para que los testículos se asienten en la parte inferior del tubo.</li>
	<li>Retire todo el heptano y la solución fijadora y enjuague los testículos rápidamente tres veces con 1x PBS.</li>
	<li>Transferir a un tubo individual limpio de 200 μL. Realizar todas las incubaciones en estos pequeños tubos para minimizar las cantidades de anticuerpos empleadas.</li>
	<li>Retire el exceso de tampón utilizando una micropipeta de 200 μL con una punta P200 y una P10 conectadas. Tenga cuidado de no aspirar testículos.</li>
	<li>Mantenga los testículos en 1x PBS sobre hielo hasta que todas las muestras estén listas.</li>
</ol>3. Tinción de anticuerpos en solución
<ol><li>Deseche el PBS y deje los testículos en PBS-T al 0,3% durante 30 min a temperatura ambiente para permeabilizar las membranas celulares.</li>
	<li>Pre-incubar en el mismo almacenador intermediario más el suero normal de la cabra del 5% (NGS) para 1 h.</li>
	<li>Añadir 200 μL del anticuerpo primario diluido en PBS-T al 0,3% más NGS al 5%(Tabla de Materiales).</li>
	<li>Incubar el anticuerpo primario durante la noche a 4 °C (o temperatura ambiente) con agitación suave en un balancín.</li>
	<li>Lavar 3 veces en PBS-T al 0,3% durante 15 min en un balancín a temperatura ambiente. Permita que los testículos se asienten en la parte inferior del tubo por gravedad.</li>
	<li>Añadir 200 μL de anticuerpo secundario diluido 1:500 de la serie conjugada DyLight.</li>
	<li>Incubar el anticuerpo secundario en una cámara oscura durante 4 h a temperatura ambiente.</li>
	<li>Lavar en PBS-T al 0,3% durante 15 min en un balancín a temperatura ambiente. Después repetir con PBS-T al 0,2% y AL 0,1% PBS-T, cada vez durante 30 min en un balancín a temperatura ambiente. Haga un lavado final en 1x PBS durante 30 min para eliminar el detergente.</li>
	<li>Añadir 180 μL de solución DAPI a 1 μg/mL en 1x PBS durante 15 min. La solución común es de 10 mg/mL enH2O. Evite hacer una solución común en PBS, que puede reducir la señal de tinción dapi en la cromatina difusa en espermatocitos más grandes.</li>
	<li>Lavar 3x durante 5 min cada uno.</li>
	<li>Utilice una pluma de barrera hidrofóbica para dibujar un círculo en un portaobjetos limpio.</li>
	<li>Aspirar los testículos y ponerlos en la diapositiva. Pre-enjuague de la punta de apertura amplia con 0.1% PBS-T evita que los testículos se peguen a la punta. Elimine el exceso de PBS.</li>
	<li>Añadir una gota (aproximadamente 100 μL) de Citifluor AF1.</li>
	<li>Coloque suavemente una cubierta de vidrio sobre el tejido, teniendo cuidado de evitar atrapar burbujas de aire.</li>
	<li>Selle la cubierta a la diapositiva con esmalte de uñas transparente.</li>
	<li>Observe los portaobjetos en un microscopio.</li>
</ol>4. Análisis de colocalización
<ol><li>Adquirir imágenes confocales. Se utilizó un microscopio confocal Zeiss LSM 710 (aceite apocromático 63x Plan) con una resolución z de 0,5 o 1 μm.</li>
	<li>Seleccione diferentes células de testículos independientes.</li>
	<li>Importe imágenes al software de procesamiento (por ejemplo, Imaris).</li>
	<li>Defina el núcleo por segmentación manual para excluir los núcleos vecinos. Utilice el software Coloc; recoge los coeficientes de correlación de Pearson (PCCs) dentro de todo el volumen entre 2 fluoróforos.</li>
</ol>

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Los autores no tienen nada que revelar.

Este protocolo proporciona un método para el immunolabeling acertado de los testículos adultos de Drosophila del entero-montaje. Como se informó en otros procedimientos, se deben usar machos jóvenes (incluso se acortó la edad a menos de 20 horas después de la eclosión) para minimizar la presencia de espermatozoides en desarrollo, lo que corre el riesgo de obstruir los espermatocitos. La disección debe ser rápida y la dilución de anticuerpos debe ajustarse para optimizar la relación señal/ruido<sup class="xref...

Los testículos de Drosophila son un modelo valioso para el estudio de la arquitectura nuclear. En las células espermatogoniales mitóticas, el volumen nuclear está ocupado en gran medida por la cromatina. Estas células se someten a cuatro rondas de división, produciendo un quiste de 16 espermatocitos primarios. Durante los próximos días, los espermatocitos pasan por 6 etapas de desarrollo (S1-S6), aumentando en gran medida en volumen, aproximadamente 20 veces1,2. También cambian su morfología nuclear. El contorno del núcleo, revelado por la lamina Dm0, se vuelve irregular y muestra profundas invaginaciones1,3. Esto se acompaña de extensos cambios en la expresión génica y modificaciones en la organización de la cromatina1,4,5,6,7. Los cromosomas interfase están bien definidos en la etapa S4-S5, formando tres masas distintas correspondientes a los 2 autosomas bivalentes principales y el par X-Y acoplado con el nucleolo.
Mad1 fue aislado originalmente como un componente clave del punto de control mitótico (revisado en8). En interfase, se describe como situado principalmente en la envoltura nuclear, sino también en el nucleoplasma9,10,11. En el testículo de Drosophila, divulgamos que Mad1 es prominente en el nucleoplasma, asociado íntimo a las dos masas de cromatina de un autosoma principales y en un grado inferior a la cromatina XY. Definimos esta estructura asociada a la cromatina como MINT (Mad1-containing IntraNuclear Territory)12. Otras cuatro proteínas se asociaron con MINTs en espermatocitos: la nucleoporina Mtor/Tpr, la SUMO peptidasa Ulp1, la proteína de punto de control mitótico Mad2 y una subunidad de un complejo similar a Polycomb Raf212.
Para terminar la descripción de MINTs, necesitábamos utilizar los anticuerpos y nos enfrentamos con los problemas técnicos encontrados generalmente con immunostaining en testículo: una permeabilidad pobre dando por resultado una no uniformidad significativa de la coloración en la profundidad del testículo, particularmente en los spermatocytes grandes. Slas preparaciones anuladas aumentaron el acceso a los anticuerpos, pero llevaron a imágenes comprimidas, restringiendo los análisis 3D y previniendo la medición de la fluorescencia cuantitativa, incluida la colocalización. El problema de la penetración de anticuerpos también podría abordarse mediante agentes permeabilizantes como el desoxicolato de Na al0,3%,pero este procedimiento no arrojó resultados reproducibles en nuestras manos. Debido a que realizamos muchos experimentos de co-etiquetado, buscamos mejorar el protocolo de permeabilización. La combinación de NP40 del 1% más heptano dio lugar a más reproducibilidad, particularmente en el estudio de los spermatocytes grandes.
Este protocolo realiza la fijación e inmunotensión de los testículos en suspensión, mejorando la calidad de la muestra, así como mejorando la reproducibilidad, y haciéndola adecuada para la microscopía confocal. Utilizamos el protocolo para definir mejor las relaciones espaciales de ciertas proteínas nucleares de la envoltura con las estructuras nucleoplásmicas de espermiocitos grandes. Este método permitirá mejores investigaciones de los cambios que acompañan el desarrollo de los espermatozoides, por ejemplo, los cambios estructurales dinámicos del núcleo, incluyendo la cromatina, el nucleoplasma y los poros nucleares.

<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

Los testículos de Drosophila son un poderoso sistema modelo para estudiar procesos biológicos, incluida la biología de células madre, la arquitectura nuclear, la meiosis y el desarrollo de espermatozoides. Sin embargo, el immunolabeling del testículo entero de Drosophila se asocia a menudo a la no uniformidad significativa de la coloración debido a la penetración del anticuerpo. Las preparaciones aplastadas solo superan parcialmente el problema, ya que disminuye la calidad 3D de los análisis. Adjunto, describimos un protocolo del entero-montaje usando NP40 y heptano durante la fijación junto con immunolabeling en medios líquidos. Conserva el volumen adecuado para la microscopía confocal junto con un etiquetado reproducible y fiable. Mostramos diferentes ejemplos de reconstitución 3D de los núcleos de espermatocitos de secciones confocales. La reproducibilidad intra e inter-testículo permite la cuantificación 3D y la comparación de la fluorescencia entre células individuales de diferentes genotipos. Se utilizaron diferentes componentes de la estructura intranuclear MINT (Mad1-que contiene intra territorio nuclear), así como dos componentes asociados con el complejo de poros nucleares para ilustrar este protocolo y sus aplicaciones en las células más grandes del testículo, los espermatocitos S4-S5.

El principal obstáculo para obtener una tinción adecuada de los testículos de Drosophila es la limitada penetrabilidad de los anticuerpos, que se ha resuelto parcialmente mediante el uso de preparaciones aplastadas pero a costa de inducir una restricción de los análisis 3D (por ejemplo, representación 3D o medida de colocalización). El procedimiento permite un etiquetado uniforme y preserva el volumen de todas las células del testículo. Aquí hemos centrado la ilustración del método en la representación 3D de...

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

Presentamos aquí un protocolo mejorado para la preparación de montaje completo y la inmunotensión de los testículos de Drosophila, adecuado para la microscopía confocal y también permitiendo el etiquetado reproducible y confiable. Ilustramos este protocolo por la representación y la cuantificación 3D de los experimentos del colocalization en los spermatocytes grandes S4-S5.

Inmunostaining De Todo El Monte Drosophila Testículos Para El Análisis Confocal 3D De Los Espermatocitos Grandes

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 vistas.  Université de Paris, F 75006, Paris, France. Presentamos aquí un protocolo mejorado para la preparación de montaje completo y la inmunotensión de los testículos de Drosophila, adecuado para la microscopía confocal y también permitiendo el etiquetado reproducible y confiable. Ilustramos este protocolo por la representación y la cuantificación 3D de los experimentos del colocalization en los spermatocytes grandes 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 de<em&gt; Drosophila</em&gt; Cámaras de huevo

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

Investigación

Biología del Desarrollo

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.

Preparación de pruebas de larvas y pupales de Drosophila para el análisis de la división celular en vivo, tejido intacto

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.

Inmunofluorescencia de ovario completo, limpieza y microscopía multifotónica para el análisis cuantitativo en 3D de la reserva ovárica en desarrollo en ratones

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.

La disección y la tinción de Drosophila Larvas Ovarios

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

Biología

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 inmunohistoquímica Abdomen

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.

Los centrosomas de imagen en Fly Testículos

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;

Análisis citológico de espermatogénesis: Los preparativos en directo y fijas de<em&gt; Drosophila</em&gt; Testículos

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.

La sonicación-facilitado Inmunofluorescencia tinción de etapa tardía embrionario y larval<em&gt; Drosophila</em&gt; Tejidos<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 de Drosophila Pupal Testis y individuales masculinos quistes línea germinal: Disección, Imaging y Tratamiento farmacológico

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.

Super-Resolución De Imágenes De Células Vivas De Estructuras Subcelulares

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

Inmunostaining De Todo El Monte Drosophila Testículos Para El Análisis Confocal 3D De Los Espermatocitos Grandes

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