这项工作是由国家科学基金会的赠款支持。

1。第1天开始之前： 健康人群中的苍蝇应保持使用标准培养协议：删除后3-4天的鸡蛋打好，并允许发展在一个恒定的温度徘徊，直到第三龄幼虫启动pupariation瓶或小瓶成人。幼虫/蛹过渡的标志是形成prepupae（0小时后，认为puparium形成，薏仁）。不动的蛹是他们的白的色彩从旧的蛹和幼虫尚未开始了他们的长椭圆形，圆润的造型和前气孔突pupariation的尊敬。 你将需要： <ul><li>一个收集蛹的油漆刷</li><li>内衬一个培养皿中培养蛹湿热试验箱：湿纸巾，滤纸收集，基因型或蛹的性别不同时间点标记</li><li> 1X磷酸盐缓冲液（PBS洗涤蛹） </li></ul>收集，培育和分期蛹<ol><li>使用湿画笔轻轻取出培养瓶/瓶0hr薏仁蛹和它们放置在湿热试验箱的盖子。 </li><li>使用画笔和1X PBS轻轻洗蛹，从蛹的情况下删除的碎片</li><li>如果需要排序按性别蛹。蛹性腺原基可用于排序的男女。男性性腺原基是一双很容易通过的约2 / 3，上下身体的长度5蛹角质层的半透明光盘的外侧。女性的性腺原基较小，不容易确定。 </li><li>蛹放置在适当标记的位置，在潮湿的室内恒温返回。文化发展到适当的时间点。 </li></ol> 2。第2天：清扫，固定和初级抗体孵育开始之前： 你将需要： <ul><li>解剖立体显微镜</li><li>手术钳</li><li>外科手术刀与数11刀片</li><li>两个9以及玻璃抑郁菜（7220-85＃康宁产品） <ul><li>填写一个菜用1X PBS井：这是冲洗清洁解剖蛹菜</li><li>第二盘与固定缓冲填充井</li></ul></li><li>湿热试验箱抗体孵育。我们使用塑料密封的夹层内衬与湿纸巾盒。 </li><li>固定缓冲液：1X PBS，4％多聚甲醛，0.2％去氧胆酸（通透） </li><li>夹层平台：坚持一方的双面胶带清除显微镜幻灯片</li><li> 1X PBS </li><li> P100或P200移液器</li></ul>解剖<ol><li>使用镊子轻轻取出一次蛹之一，他们面临的最广泛的磁带宽度蛹的前端放在夹层平台。如果蛹过分潮湿，印迹他们干用纸巾。 </li><li>蛹之前已经完全坚持到磁带上，用画笔位置。 </li><li>允许蛹空气干燥，并坚持到磁带（5-10分钟） </li><li>与外科手术刀解剖双边每个蛹。前蛹后用一个单一的快速削减，这是最好的。如果正确完成，削减将平分前部和后部的气孔对。剖析作为洗涤和清洁的样品迅速是必要的，以防止蛋白水解损害组织的时间不超过10蛹。 </li><li>使用画笔，转让少量的1X PBS每个解剖蛹放松他们从磁带。 </li></ol>清洁，固定和初级抗体孵育<ol start="6"><li>随着对手术钳抓住个别蛹一半前方和立即沉浸在一个良好的清洗盘。应改为显微镜舞台上的漂洗菜夹层平台。蛹的情况下，应保留的样本，直到处理完成后，和样品准备安装。 </li><li>虽然仍然抓蛹一半使用移液器轻轻洗去腹部的内部组织。在清洗过程中的太多的压力也可导致上皮细胞的损失。 </li><li>立即转移清洁蛹一半固定缓冲液在室温下，同样的过程中剩余的蛹半。应采取与实践，清扫和清洗20蛹半不到5分钟。 </li><li>允许蛹到固定缓冲区为1（一）小时，在室温下孵育。 </li><li>在1X PBS冲洗固定蛹3X五分钟</li><li>样品可立即进行免疫组化处理，或可存储100％的乙醇在-20 ° C至3个月，不丧失的细胞或抗原表位反应。 </li><li>要储存样品，平衡通过稀释样品PBS的系列：在室温为20分钟，每个稀释度的温度前，转移到100％乙醇的乙醇（3:1，1:1，1:3）。 </li><li>样品必须重新平衡通过反向系列免疫组织化学处理前的PBS。 </li><li>除了初级抗体之前阻止样品1（一）小时1X PBS（2％牛血清白蛋白补充）。 </li><li>孵育抗体在夜间在1X PBS稀释至适当浓度4 °没有摇摆彗星。 </li></ol> 3。第3天：中抗体孵化，安装和成像开始之前： 你将需要： <ul><li>两个手术钳</li><li>安装介质（甘油，矢量Vectashield [实验室]，或Slowfade黄金[Invitrogen公司]） </li><li>萧条以及显微镜载玻片（0.8毫米） </li><li>盖玻片（24 × 40毫米） </li></ul>二抗孵育和安装： <ol><li>洗净样品3X 10分钟，用1X PBS </li><li>此外之前的二级抗体阻止样品1（一）小时1X PBS（2％的牛血清白蛋白补充）。 </li><li>孵育二级抗体在室温下在黑暗中没有适当的浓度在1X PBS稀释为3（3）小时摇摆。 </li><li>洗净样品3X 10分钟，用1X PBS </li><li>如果合适，染液蛹（示例我们的DAPI核染色[4&#39;,6 - diamidino - 2 -苯基吲哚] PBS稀释10分钟）。 </li><li>在安装前的适当媒体平衡的样本（至少30分钟）。 </li><li>准备一个样本幻灯片。蛹的形态，防止平面安装。抑郁症幻灯片成像（下降幻灯片）以及安装在样品。在抑郁症以及安装媒体广场75 - 100ul。一些样品，可安装在一个单一的幻灯片。 </li><li>蛹的情况下，必须从样本中删除后再安装。抓住个别蛹的情况下，前方确保不掌握内部蛹膜。 </li><li>随着第二对镊子轻轻抓住头内部的蛹膜，从蛹的情况下删除。 </li><li>立即将样本的抑郁症，并继续处理额外的样品。 </li><li>使用探针或镊子的位置，样品中的抑郁症以及侧面朝上均匀。偶尔的气泡可以用探针移除。 </li><li>轻轻地照顾到的样品盖玻片不引入气泡</li></ol> 4。代表性的成果： 样品准备使用该协议保留的成年人腹部的大体形态。可投射图像堆栈生成一个二维图像或3 - D渲染，可应用于腹部拓扑调查。 <img src="/files/ftp_upload/3139/3139fig1.jpg" alt="图1"/> 图1。与鼠抗- WG（4D4：爱荷华州的杂交瘤细胞银行）和抗GFP 基因在果蝇蛹的分割产品无翅的蛋白质和Engrailed表达（EN - GAL4 UAS - GFP）。puparium形成后的26小时（APF）的可视化。 10倍的放大倍率，前左和背。 Nucleii counterstained用DAPI。 成堆的约50幅图像（ΔZ之间切片2.5μm）预计。

<ol>
	<li>Kopp, A., Duncan, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anteroposterior+patterning+in+adult+abdominal+segments+of+Drosophila.">Anteroposterior patterning in adult abdominal segments of Drosophila.</a> Dev. Biol. 242, 15-30 (2002).</li><li>Ninov, N., Chiarelli, D. A., Martin-Blanco, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrinsic+and+intrinsic+mechanisms+directing+epithelial+cell+sheet+replacement+during+Drosophila+metamorphosis.">Extrinsic and intrinsic mechanisms directing epithelial cell sheet replacement during Drosophila metamorphosis.</a> Development. 134, 367-379 (2007).</li><li>Bischoff, M., Cseresnyes, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+rearrangements,+cell+divisions+and+cell+death+in+a+migrating+epithelial+sheet+in+the+abdomen+of+Drosophila.">Cell rearrangements, cell divisions and cell death in a migrating epithelial sheet in the abdomen of Drosophila.</a> Development. 136, 2403-2411 (2009).</li><li>Matsuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+Morphology+and+evolution+of+the+insect+abdomen.">in Morphology and evolution of the insect abdomen.</a> , (1976).</li><li>Kerkis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Growth+of+the+Gonads+in+Drosophila+Melanogaster.">The Growth of the Gonads in Drosophila Melanogaster.</a> Genetics. 16, 212-224 (1931).</li></ol>

没有利益冲突的声明。

在这个视频中介绍的技术可以被用来从果蝇蛹的发育时间点的各种准备。蛹处理，32小时在24小时内薏仁薏仁是最容易从上皮细胞的损失。延长孵化步骤的过程中使用的洗涤剂（如TRITON X - 100和Tween - 20）增加细胞的损失的可能性，因此不推荐。相反，去氧胆酸是用来作为洗涤剂，在最初的固定步骤。在1X PBS不用洗衣粉的所有后续步骤进行。此外，摇摆样本期间延长孵化步骤，增加细胞的损失，应当避免。 样品可以利用共聚焦显微镜技术成像。然而， 果蝇蛹处理，也可以使用结构照明显微镜系统产生的图像质量相媲美共聚焦技术成像。

<ul><li>解剖立体显微镜</li><li>手术钳</li><li>外科手术刀与数11刀片</li><li>九井玻璃抑郁菜（康宁产品＃7220-85） </li><li>培养蛹湿热试验箱</li><li> 1X磷酸盐缓冲液（PBS） </li><li> ＃11外科手术刀</li><li>双面胶带</li><li>固定缓冲液：1X PBS，4％多聚甲醛，0.2％去氧胆酸（通透） </li><li> P100或P200移液器</li><li>萧条以及显微镜载玻片（0.8毫米） </li></ul>

drosophila pupal abdomen immunohistochemistry

果蝇蛹腹部为上皮细胞的形态发生和发展 1-3性二态形态研究建立的模型系统。在化蛹，跨度约96小时（在25 ° C），成虫细胞增殖人口取代幼虫表皮产生成人腹部分部。这些成虫细胞，胚胎发育过程中出生的，存在对每个幼虫腹节侧histoblast巢。四双histoblast巢会引起成年背角质层（前部和后部的背巢），腹面角质层（腹侧巢），并与各分部（气门巢 ）4相关的气孔。 puparation后，这些二倍体细胞（按大小区分多倍体幼虫的表皮细胞，晶状体上皮细胞）开始的晶状体上皮细胞的增殖，迁移和更换的陈旧观念的过程。可以采用不同的分子和遗传工具，调查涉及成人的腹部形态发生的遗传途径的贡献。极致的成人表型是典型的分析成人​​腹部表皮以下的夹层。然而，调查的基本分子过程需要蛹上皮细胞的免疫组织化学分析，这为我们带来了独一无二的的挑战。时间上的动态形态发生和两个不同的上皮人口（幼虫和成虫）的相互作用生成一个脆弱的组织，容易清扫和后续处理过程中细胞过度损失。我们已经制定了清扫，固定，安装和果蝇蛹abdominem上皮细胞生成适合焦或标准的荧光显微镜的一贯的高品质样本的免疫组织化学研究的成像方法。

Watch this Scientific Journal Video about Drosophila Pupal Abdomen Immunohistochemistry at JoVE.com

Drosophila Pupal Abdomen Immunohistochemistry

抗体染色<em&gt;果蝇</em&gt;蛹可以提高成人腹部发育遗传学的遗传分析。我们目前对我们的协议清扫，固定和抗体染色上演<em&gt;果蝇</em&gt;蛹腹部。

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 ...

drosophila-pupal-abdomen-immunohistochemistry

Research

JoVE Journal

Biology

Drosophila Pupal Abdomen Immunohistochemistry

16.1K 次观看.  University of Alabama. 抗体染色果蝇蛹可以提高成人腹部发育遗传学的遗传分析。我们目前对我们的协议清扫，固定和抗体染色上演果蝇蛹腹部。

视频: Drosophila Pupal Abdomen Immunohistochemistry

Thin Sectioning of Slice Preparations for Immunohistochemistry

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been preserved, freshly removed, or excised but kept viable, as in slice preparations of brain tissue.  Subsequent microscopic studies of this material can be challenging, as the tissue samples may be difficult to handle.  Demonstrated here is a method for obtaining thin cryostat sections of tissue with a thickness that may range from 0.2-5.0 mm.  We routinely cut 400 micron thick Vibratome brain slices serially into 5-10 micron coronal cryostat sections.  The slices are typically first used for electrophysiology experiments and then require microscopic analysis of the cytoarchitecture of the region from which the recordings were observed.  We have constructed a simple device that allows controlled and reproducible preparation and positioning of the tissue slice.  This device consists of a cylinder 5 cm in length with a diameter of 1.2 cm, which serves as a freezing stage for the slice.  A ring snugly slides over the cylinder providing walls around the slice allowing the tissue to be immersed in freezing compound (e.g., OCT).  This is then quickly frozen with crushed dry ice and the resulting  wafer  can be position easily for cryostat sectioning.  Thin sections can be thaw-mounted onto coated slides to allow further studies to be performed, such as various staining methods, in situ hybridization, or immunohistochemistry, as demonstrated here.

The present method allows reproducible cryostat sectioning of small, difficult-to-manage, tissue pieces, such as biopsies and brain slices. We utilize a simple aluminum freezing stage to facilitate handling of tissue and a standard cryostat to routinely produce 5-10 micron serial sections from 400 micron thick brain slices.

薄切片进行免疫组化切片的筹备工作

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been ...

科研

生物学

In situ Protocol for Butterfly Pupal Wings Using Riboprobes

Here we present, in video format, a protocol for in situ hybridizations in pupal wings of the butterfly Bicyclus anynana using riboprobes.  In situ hybridizations, a mainstay of developmental biology, are useful to study the spatial and temporal patterns of gene expression in developing tissues at the level of transcription. If antibodies that target the protein products of gene transcription have not yet been developed, and/or there are multiple gene copies of a particular protein in the genome that cannot be differentiated using available antibodies, in situs can be used instead. While an in situ technique for larval wing discs has been available to the butterfly community for several years, the current protocol has been optimized for the larger and more fragile pupal wings.

In order to examine gene expression in the pupal wing tissue of Bicyclus anynana, we present an optimized protocol for in situ hybridizations using riboprobes. We also provide guidelines for the further optimization of this protocol for use in pupal wings of other Lepidopteran species.

在原位议定书“蝴蝶蛹使用Riboprobes翼

Here we present, in video format, a protocol for in situ hybridizations in pupal wings of the butterfly Bicyclus anynana using riboprobes.  In situ ...

Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.

免疫组化：石蜡切片从Vector实验室使用Vectastain ABC试剂盒

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific ...

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, suprabasal, and stratum corneum layers. The precise localization within the epidermis of a particular protein lends clues toward its functional role within the epidermis. In this chapter, we describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory. Paraffin sections generally provide much better morphology, hence, superior results and photographs; however, not all antibodies will work with the harsh fixation and treatment involved in their processing. Therefore, the protocol for frozen sectioning is also included. Within paraffin sectioning, two fixation protocols are described (Bouin's and paraformaldehyde); the choice of fixative will be directly related to the antibody specifications and may require another fixing method.

We describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory.

使用荧光抗体对小鼠表皮石蜡切片的免疫组织化学

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, ...

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

果蝇幼虫NMJ免疫

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas

The tissue microarray (TMA) technology provides the means for high-throughput analysis of multiple tissues and cells. The technique is used within the Human Protein Atlas project for global analysis of protein expression patterns in normal human tissues, cancer and cell lines. Here we present the assembly of 1 mm cores, retrieved from microscopically selected representative tissues, into a single recipient TMA block. The number and size of cores in a TMA block can be varied from approximately forty 2 mm cores to hundreds of 0.6 mm cores. The advantage of using TMA technology is that large amount of data can rapidly be obtained using a single immunostaining protocol to avoid experimental variability. Importantly, only limited amount of scarce tissue is needed, which allows for the analysis of large patient cohorts 1 2. Approximately 250 consecutive sections (4 &mu;m thick) can be cut from a TMA block and used for immunohistochemical staining to determine specific protein expression patterns for 250 different antibodies. In the Human Protein Atlas project, antibodies are generated towards all human proteins and used to acquire corresponding protein profiles in both normal human tissues from 144 individuals and cancer tissues from 216 different patients, representing the 20 most common forms of human cancer. Immunohistochemically stained TMA sections on glass slides are scanned to create high-resolution images from which pathologists can interpret and annotate the outcome of immunohistochemistry. Images together with corresponding pathology-based annotation data are made publically available for the research community through the Human Protein Atlas portal (<a href="http://www.proteinatlas.org" target="_blank">www.proteinatlas.org</a>) (Figure 1) 3 4. The Human Protein Atlas provides a map showing the distribution and relative abundance of proteins in the human body. The current version contains over 11 million images with protein expression data for 12.238 unique proteins, corresponding to more than 61% of all proteins encoded by the human genome.

Tissue microarrays allows for an efficient method to gain concurrent information from a multitude of tissues. Representative parts of tissues are assembled into a single paraffin block. Sections from the block are used for immunohistochemistry and analysis of protein expression patterns. Digital scanning generates corresponding images for distribution of data.

组织芯片，​​免疫组织化学染色和数字化生产在人类蛋白质图集

The  tissue microarray (TMA) technology provides the means for high-throughput  analysis of multiple tissues and cells. The technique is used within the ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

视网膜平片准备成像和免疫组化优化方案

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

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 ...

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 ...