作者要感谢晃千叶的智力支持，物质支持和股票。感谢朱莉娅Dallman征求意见。

1。飞工作<ol><li>保持苍蝇在室温下10个标准玉米面-琼脂糖蜜酵母培养基。 </li><li>对于十字架，隔离在6小时羽化的处女。穿越到所需基因型的男性后，改变飞行到新瓶每3-4天。 </li></ol>注：对于这些实验，Gal4的线A9被用来驱动转基因的表达在边路。飞股份与股票中心布卢明顿获得。在这些实验中使用的股票包括A9-GAL4（BL＃8761），His2Av-GFP（BL＃5941），在sco / CYO HsCre（BL＃1092），UAS-ChRFP-浴池（BL＃25773），lollibow 11。 2。选择和蛹的安装<ol><li>蛹阶段无论是通过收集它们作为白色预蛹（WPP），保证它们在25℃，直至达到适当的年龄或使用形态学标准12。 </li><li>观察细胞分裂，选择蛹最近经历头外翻。从这时起，直到刚刚羽化（在&gt; 96小时）之前，蛹是不动的，允许延长时间推移观察。 </li><li>先接触他们用沾有水的画笔，等待一分钟，让水松粘合，然后轻轻地督促他们到画笔从瓶中取出蛹。 </li><li>轻轻地用刺戳它们与在水中的画笔除去唾液腺粘合剂和食物颗粒洗选定蛹。 </li><li>转让清洁蛹到25毫米培养皿盖玻片底（数量1个半盖玻片）。 </li><li>采用细条或者橡皮泥或牙科用蜡作为支撑的，安装蛹，让感兴趣的组织是最接近盖玻片。 注意：在这里描述的研究，蛹翅膀，脚，腹部histoblasts或背侧notum已经观察到。在实践中，在20微米蛹壳的表面上的任何组织的观察。 </li><li>东方蛹小心，这样的布甲电子商务的兴趣是平行于玻璃盖玻片表面。 </li><li>一旦蛹安装，使用画笔来一层薄薄的硫代二甘醇（TDG）的转移到蛹和盖玻片之间的空间。 </li></ol>注意：TDG减少表面散射，石油的折射率相匹配，并允许氧气渗入组织13。使用油不建议，因为他们往往​​剥夺氧气的组织，引起细胞行为的迅速停止。 3。成像注意：对于成像，共焦显微镜很可能是必要的，因为在共焦除去大部分遮蔽背景所造成的蛹壳的强烈照明。 <ol><li>调整的共聚焦设置以最小化照明对蛹发育和存活的影响。要做到这一点，罢工的激励强度和收集的敏感性之间的平衡。成像无线的典型配置第徕卡SP5中使用的共振扫描器（8000赫兹），激光功率设定为2％（20％的功率的10％透射率），针孔设定为120微米，行平均设置为8。设置将取决于实验条件而变化。 </li></ol>注意：要解决精细尺度特征40X和63X油浸镜头（0.1毫米的工作距离）也已成功应用于这种方法，虽然他们限制焦深。 4。分析为了分析框架，Z-堆栈或电影导入数据文件到斐济，其中有有效的工具来查看，测量和修改文件简报14。例如时间完成有丝分裂使用采样间隔和多维图像浏览器来浏览帧一边看从前期的细胞，以末期进行测定。 x轴，y-和z-维度可使用测量工具来测量。有关软件及其使用的详细说明见：http://fiji.sc/Fiji。

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作者什么都没有透露。

可视化，测量和量化分裂细胞的功能，用于通过His2AvGFP表达细胞共聚焦分析的方法观察有丝分裂的果蝇蛹生活机翼的简单准备所需的开发。这种方法被用来记录该细胞周期中的蛹翼熊强的相似性对细胞周期的假复层上皮细胞中的细胞核转移到他们进入有丝分裂的上皮细胞的顶面。以下末期，核回落到上皮细胞层。因此，这种单层，显然不分层上皮细胞，显示的单元格的限制interkinetic核迁移?...

<html>醋蝇， 果蝇 ，是一种行之有效的模型用于研究生物学的许多方面。 果蝇的研究具有遗传实验的丰富的历史，使基因操作的复杂形式，包括表达，敲除和基因突变。同的荧光蛋白标记的出现，这个曲目已经扩大到包括细胞和蛋白质的研究在活的动物。在果蝇胚胎是一个很好的系统，这样的研究，因为它是小，光学透明的，允许深，高分辨率成像在体内 1-3。飞发展的其他阶段已被证明是易于处理的少，需要麻醉4，解剖和短期培养5,6或增设在角质层成像7,8窗口。这些操作通常是妥协的动物发展的长期或影响动物的方式，限制了成像时间很短。 ENT“&gt;为了研究新的突变基因中像细胞周期调控，有必要找到一个适当的准备，以研究细胞周期的时序和保真度。由于大多数胚胎细胞周期截断（SM或S-G2-M）和所研究的突变体没有表现出缺陷，直到后期，观察细胞周期中蛹期的组织是很重要的。上皮细胞在蛹有一个更典型的G1-S-G2-M期细胞周期和现阶段的蛹是不能够肌肉运动9。初始起点操作包含完整的整体蛹表达Histone2AV-GFP。尽管蛹壳的表观不透明度，这完好制剂被证明是优良的长期的体内成像，这种技术是简单以至于本科生科研人员经常使用它来 ​​研究细胞和发育生物学果蝇的方面，但分辨率是好的足以让的微米尺度特征歧视。用这种方法，过小时，分钟或秒事件的观察是可能简单地通过调节时间序列的参数。使用蓝色，绿色，黄色，橙色，和红色荧光蛋白，或这些的组合的视频，已经作出。重要的是，如果小心，尽量减少激光强度，甚至长期成像对发展的动物或生存没有影响。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fly stuff fly pad</td>
			<td style="width:71px;">Genesee scientific</td>
			<td style="width:119px;">59-114</td>
			<td style="width:167px;">for fly anesthetization</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CO2 gas</td>
			<td style="width:71px;">Airgas south</td>
			<td style="width:119px;">CD50</td>
			<td>For fly anesthetization</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Regulator</td>
			<td style="width:71px;">Airgas south</td>
			<td style="width:119px;"> </td>
			<td>CO2 regulator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fly vials</td>
			<td style="width:71px;">Genesee scientific</td>
			<td>32-113RLBF</td>
			<td>Fly culture</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Drosophila lines:A9-Gal4 (Bl#8761), His2Av-GFP (Bl#5941), Sco/CyO HsCre (Bl#1092), UAS-ChRFP-Tub (Bl#25773)</td>
			<td style="width:71px;">Bloomington Stock center</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass bottom dishes #1 1/2</td>
			<td style="width:71px;">WillCo Wells BV</td>
			<td style="width:119px;"> </td>
			<td>For microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Thiodiethylene Glycol</td>
			<td style="width:71px;">Fluka</td>
			<td align="right" style="width:119px;">88559</td>
			<td>mountant</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Modeling clay</td>
			<td style="width:71px;">art supply store</td>
			<td style="width:119px;"> </td>
			<td>Support to position pupae against</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Paintbrushes</td>
			<td style="width:71px;">art supply store</td>
			<td style="width:119px;"> </td>
			<td>To manipulate flies</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Fine Forceps, Inox #5</td>
			<td style="width:71px;">Fine science tools</td>
			<td style="width:119px;">11252-20</td>
			<td>Dumont #5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">computer</td>
			<td style="width:71px;">any</td>
			<td style="width:119px;"> </td>
			<td>8 Gb RAM for image/movie analysis</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:216px;">Fiji software</td>
			<td style="width:71px;"> </td>
			<td style="width:119px;">Free ware http://fiji.sc/Fiji</td>
			<td>Image analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Confocal microscope</td>
			<td style="width:71px;"> </td>
			<td style="width:119px;"> </td>
			<td>Any fast scanning confocal should be sufficient</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">20x dry, and 40x or 63x oil immersion lenses</td>
			<td style="width:71px;">any</td>
			<td style="width:119px;"> </td>
			<td>For imaging tissue, cellular and subcellular features</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immersion oil (non fluorescent)</td>
			<td style="width:71px;"> </td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereo microscope</td>
			<td style="width:71px;">any</td>
			<td style="width:119px;"> </td>
			<td>For fly manipulation</td>
		</tr>
	</tbody>
</table>

imaging through the pupal case of drosophila melanogaster

长期以来利用果蝇作为模型细胞和发育生物学取得了一系列工具。总之，这些技术已成功地从各种方法论的角度细胞和发育生物学的分析。实时成像是动态观察细胞过程，如细胞分裂或细胞运动的一个新兴的方法。经分离的突变未知推测细胞周期蛋白成为必不可少使用实时成像，观察有丝分裂原位 。在果蝇中最实时成像研究都集中在那些由于它们的小尺寸和光学清晰度访问操作和观察的萌芽阶段。然而，在这些阶段的细胞周期是不寻常的，因为它缺少一个间隙阶段或两者。相比之下， 果蝇蛹翼的细胞具有典型的细胞周期并经过一段快速的有丝分裂横跨约20小时，蛹的发展。很容易我找出并隔离适当阶段蛹赶在有丝分裂原 。安装完好蛹成像过程中提供易处理性和耐用性的最佳组合，使实验来进行数小时对细胞和动物存活率的影响降至最低。该方法允许观测的特征一样小，或小于飞染色体。的显微镜设置和安装的细节调整，允许延长制剂的可视化相邻小区的膜动力学和荧光标记的蛋白，如微管蛋白。此方法适用于所有测试的荧光蛋白，可以在各种时间尺度的把握亚微米尺度特征。虽然仅限于外20微米的蛹与传统的共聚焦显微镜，这种方法观察蛋白质和细胞动力学在蛹体内的组织一般可有用细胞和发育生物学在这些组织的研究。

细胞的假复层上皮，如显影果蝇眼睛，或显影脊椎动物中枢神经系统的心室层，经历核运动，称为interkinetic核迁移，在时间上与细胞周期。当核是在或接近基底表面和细胞进入有丝分裂时细胞核到达顶端面15,16发生DNA的复制。在头外翻后的前几个小时，蛹翼细胞形成一个快速分裂的单层上皮。收集在XYZT模式的数据从早期蛹翼，采取每节2微米以1分钟的间隔。在所得到的3D电影有丝分?...

Watch this Scientific Journal Video about Imaging Through the Pupal Case of Drosophila melanogaster at JoVE.com

Imaging Through the Pupal Case of Drosophila melanogaster

本文演示了如何使用直接通过蛹壳快速扫描共聚焦显微镜对细胞图像的行为。将其留在蛹壳完好无损，此方法允许观察和动态细胞过程的测量在果蝇发育的阶段，是很难直接学习。

通过影像学的蛹壳<em&gt;果蝇</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

imaging-through-the-pupal-case-of-drosophila-melanogaster

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JoVE Journal

Biology

Imaging Through the Pupal Case of Drosophila melanogaster

13.3K 次观看.  University of Miami. 本文演示了如何使用直接通过蛹壳快速扫描共聚焦显微镜对细胞图像的行为。将其留在蛹壳完好无损，此方法允许观察和动态细胞过程的测量在果蝇发育的阶段，是很难直接学习。 

视频: Imaging Through the Pupal Case of Drosophila melanogaster

Dissection of Larval CNS in Drosophila Melanogaster

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

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

在果蝇幼虫中枢神经系统的剖析

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

科研

生物学

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

实时成像果蝇胚胎血细胞迁移

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

直立成像果蝇胚胎

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

从成人Oenocytes剖析果蝇</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

分离果蝇睾丸

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

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.

果蝇蛹腹部免疫

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

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

寿命测量<em&gt;果蝇</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

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.

解剖，成像和药物治疗： 果蝇蛹睾丸和单身男性生殖系囊肿的体外培养

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

Dissection and Mounting of Drosophila Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

解剖和安装的<em&gt;果蝇</em&gt;蛹眼光盘

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...

Maintenance of a Drosophila melanogaster Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

的维持<em&gt;果蝇</em&gt;人口笼

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...