我们感谢P.韦里泽，J.约翰森H.，奥库拉的抗体和A.德贝克，布卢明顿和维也纳的苍蝇库存中心。感谢杰克逊的建议和刺激的讨论。

1. 德罗索菲拉睾丸收藏
注:年轻男性（0-15小时后关闭）是检查双体生殖细胞（即精子卵细胞和精子细胞）所必需的。
<ol><li>尽可能多地选择幼蝇，以尽量减少来自发育精子的干扰。</li>
	<li>麻醉苍蝇通过短暂接触二氧化碳和转移到飞垫14，15。</li>
	<li>在解剖显微镜（10倍放大），使用钳子去除头部。</li>
	<li>将 5 到 10 只被斩首的苍蝇转移到 100 μL 的林格缓冲区（183 mM KCI、47 mM NaCl、10 mM Tris-HCI、1 mM EDTA、完整的蛋白质抑制剂、pH 6.8）在黑色背景支撑下的硅胶涂层玻璃滑梯上。</li>
	<li>解剖显微镜的放大倍数为40倍，使用一对5号钳子在胸腔和腹部之间保持苍蝇，并用其他钳子将腹部从胸腔拉开。迅速将睾丸和相邻组织从飞尸体15中分离出来，并在同一滑梯上放置一滴新的林格缓冲器。</li>
	<li>所有睾丸准备好后，从剩余的组织（辅助腺体和外生殖器）中清洁睾丸。</li>
	<li>从包含解剖睾丸的滴中取出多余的缓冲区。通常，使用 p200 移液器，在 200μL 尖端顶部安装 10 μL 尖端。这允许通过一个小孔径去除多余的液体。将4%的甲醛固定在含有解剖睾丸的液滴上，然后迅速将睾丸转移到含有新鲜固定溶液的管子上。</li>
</ol>2. 甲醛固定
<ol><li>使用前准备 2 mL 的固定溶液:在磷酸盐缓冲盐水 （PBS） 中稀释 4% 副甲醛的 600 μL，其中 30 μL 的 NP40 20% 加 1200 μL 的乙烷在 2 mL 硅化微中微富格管中。在 600μL 单环中使用 4% 的 PBS 中副甲醛，储存在 -20°C。 注意:甲烷和甲醛是有毒的，应在烟灰罩中处理。按照主办机构的规定处理。</li>
	<li>使用带宽孔径尖的 p200 微管将睾丸转移到 PFA/NP40/heptane 解决方案。用手上下摇动管子30s。在室温下继续将睾丸固定在旋转搅拌机上 30 分钟。</li>
	<li>停止旋转搅拌机，将管子放在管架中，以便相分离，并让睾丸沉淀到管的底部。</li>
	<li>取出所有六烷和固定溶液，用 1 倍 PBS 快速冲洗睾丸三次。</li>
	<li>转移到一个干净的200μL单管。在这些小管中执行所有孵化，以最大限度地减少所使用的抗体量。</li>
	<li>使用附带 P200 和 P10 提示的 200μL 微皮带去除多余的缓冲区。小心不要吸气睾丸。</li>
	<li>将睾丸放在冰上1x PBS中，直到所有样品都准备好。</li>
</ol>3. 溶液中的抗体染色
<ol><li>丢弃 PBS，在室温下将睾丸留在 0.3% PBS-T 中 30 分钟，以渗透细胞膜。</li>
	<li>在同一缓冲区预孵化，加上5%的正常山羊血清（NGS）1小时。</li>
	<li>在 0.3% PBS-T 加上 5% NGS （材料表） 中稀释的主要抗体中加入 200 μL。</li>
	<li>在 4 °C（或室温）下在夜间孵化主要抗体，在摇杆上轻轻搅拌。</li>
	<li>在室温下在摇动器上用 0.3% PBS-T 清洗 3 次 15 分钟。允许睾丸按重力沉降到管子底部。</li>
	<li>从 DyLight 组合系列中添加 200 μL 的二次抗体稀释 1:500。</li>
	<li>在室温下在暗室中孵育二次抗体4小时。</li>
	<li>在室温下在摇动器上清洗 0.3% PBS-T 15 分钟。然后重复0.2%PBS-T和0.1%PBS-T，每次在室温下在摇动器上30分钟。在 1 倍 PBS 中进行 30 分钟的最终洗涤，以去除洗涤剂。</li>
	<li>在 1 倍 PBS 中以 1μg/mL 的速度加入 180μL 的 DAPI 解决方案，15 分钟。库存溶液为 H 2 O 中的10毫克/mL，避免在 PBS 中制作库存溶液，从而减少较大精子细胞中扩散染色质上的 DAPI 染色信号。</li>
	<li>每次洗3次5分钟。</li>
	<li>使用疏水屏障笔在干净的滑梯上画一个圆圈。</li>
	<li>吸气睾丸，并把它们放在幻灯片上。用 0.1% PBS-T 预冲洗宽孔径尖可防止睾丸粘在尖端上。删除多余的 PBS。</li>
	<li>添加一滴（约100μL）的花旗流体AF1。</li>
	<li>轻轻将玻璃盖在纸巾上，小心避免诱捕气泡。</li>
	<li>使用透明指甲油将盖片密封在滑梯上。</li>
	<li>观察显微镜上的幻灯片。</li>
</ol>4. 同地分析
<ol><li>获取对焦图像。我们使用了蔡司 LSM 710 共焦显微镜 （63x 计划杏色油）， z 分辨率为 0.5 或 1 μm。</li>
	<li>从独立睾丸中选择不同的细胞。</li>
	<li>将图像导入到处理软件（例如，Imaris）。</li>
	<li>通过手动分割来定义原子核，以排除相邻的核。使用科洛克软件;它收集皮尔逊的相关系数（PCCs）在整个体积之间的2个氟磷。</li>
</ol>

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作者没有什么可透露的。

该协议为成功免疫全安装成人嗜血杆菌睾丸提供了一种方法。正如在其他程序中报告，年轻男性必须使用（我们甚至缩短年龄到20小时后，关闭），以尽量减少发展精子的存在，这有可能阻碍精子细胞。解剖应快速进行，抗体稀释必须进行调整，以优化信号与噪声比21、22、23。因为使用其他协议，我们没有观察到整个睾丸深度?...

果蝇睾丸是研究核建筑的宝贵模型。在线粒体精子癌细胞中，核体积主要被色质所占据。这些细胞经过四轮分裂，产生16个原发性精子细胞的囊肿。在接下来的几天里，精子细胞通过6个发育阶段（S1-S6），体积大大增加，大约是20倍1，2。它们也改变了其核形态。层压 Dm0 揭示的原子核轮廓变得不规则，并显示深层入侵1，3。伴随着广泛的基因表达变化和改变染色质组织1，4，5，6，7。相间染色体在S4-S5阶段定义明确，形成三个不同的质量，对应于2个主要双价自体体和与核对接的X-Y对。
Mad1 最初被隔离为线粒体检查点的关键部分（在8中审查）。在相间，它被描述为主要位于核信封，但也在核质9，10，11。在嗜血杆菌睾丸中，我们报告说，Mad1在核质中很突出，与两种主要的自体染色质质量密切相关，在较小程度上与XY色素有关。我们定义这种染色质相关结构为MINT（含Mad1核内领土）12。其他四种蛋白质与精子细胞中的MINT相关:核苷Mtor/Tpr、SUMO肽酶乌尔普1、线粒体检查点蛋白Mad2和类似聚团的复合Raf212的亚单位。
为了完成对MINT的描述，我们需要使用抗体，并面临着睾丸中免疫稳定通常遇到的技术问题:渗透性差，导致睾丸深度的污渍严重不均匀，特别是在 大型精子细胞中。S消解制剂 增加了抗体访问，但 导致压缩图像，限制3D分析， 并 防止定量荧光的测量 ，包括同位素化。抗体渗透的问题也可以通过渗透剂，如0.3%纳脱氧色素13来解决，但这个程序并没有在我们手中产生可重复的结果。因为我们进行了许多联合标记实验，我们期待改进渗透协议。1% NP40 加希普坦的组合导致更多的可重复性，特别是在研究大型精子细胞方面。
本协议对悬浮睾丸进行固定和免疫维护，提高样品质量，提高可重复性，使其适合共聚焦显微镜。我们利用该协议更好地定义某些核包络蛋白与大型精子细胞核质结构的空间关系。这种方法将允许更好地研究伴随精子细胞发育的变化，例如细胞核的动态结构变化，包括色素、核质和核毛孔。

<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

嗜血 性睾丸是研究生物过程的强大模型系统，包括干细胞生物学、核结构、肌瘤和精子发育。然而，由于抗体渗透，整个 嗜血杆菌 睾丸的免疫带通常与染色的严重不均匀性有关。压扁的准备工作只是部分克服了这个问题，因为它降低了分析的3D质量。在此，我们描述了使用NP40和六烷在固定过程中与液体介质免疫带的全安装协议。它保留了适合共焦显微镜的体积以及可重复和可靠的标签。我们展示了不同的例子，从共聚焦部分的精子细胞核的3D重组。睾丸内部和睾丸间可重复性允许不同基因型的单个细胞之间的荧 光 进行3D定量和比较。我们使用了核内造币厂结构（含Mad1的核领土内部）的不同组件以及与核孔复合物相关的两个组件来说明该协议及其在睾丸最大细胞S4-S5精子细胞上的应用。

获得充分染色的Drosophila睾丸的主要障碍是抗体的渗透性有限，通过使用压扁制剂部分解决，但代价是导致对3D分析的限制（例如3D表示或同地测量）。该程序允许统一标记，并保留睾丸所有细胞的体积。在这里，我们重点介绍了S4-S5精子细胞的3D表示方法，因为它们是最大的细胞，因此对挤压变形更敏感。我们使用了不同的核标记，包括核苷酸和核内薄荷域（含Mad1核领土）的标签，由线粒体蛋白Mad...

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

我们在这里提出了一个改进的协议，为全安装准备和免疫染色的 嗜血杆菌 睾丸，适合共焦显微镜，也允许可重复和可靠的标签。我们通过对大型S4-S5精子细胞的同位素化实验进行3D表示和量化来说明此协议。

用于大型精子细胞三维共焦分析的全山 嗜血杆菌 睾丸免疫染色

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 次观看.  Université de Paris, F 75006, Paris, France. 我们在这里提出了一个改进的协议，为全安装准备和免疫染色的 嗜血杆菌 睾丸，适合共焦显微镜，也允许可重复和可靠的标签。我们通过对大型S4-S5精子细胞的同位素化实验进行3D表示和量化来说明此协议。

视频: 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.

直立成像<em&gt;果蝇</em&gt;蛋钱伯斯

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

科研

发育生物学

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.

用于活、完整组织中细胞分部分析的果蝇拉瓦尔和普帕尔睾试验的制备

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.

全卵巢免疫荧光、清除和多光子显微镜用于小鼠发育中卵巢储备的定量3D分析

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.

夹层和染色果蝇幼虫卵巢

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

生物学

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 ...

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.

在飞睾丸成像中心体

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;

精子发生的细胞学分析：Live和固定制剂<em&gt;果蝇</em&gt;睾丸

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.

后期阶段的胚胎和幼虫的超声，促进免疫荧光染色<em&gt;果蝇</em&gt;组织<em&gt;原位</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.

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 ...

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.

亚细胞结构的超分辨率活细胞成像

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

用于大型精子细胞三维共焦分析的全山嗜血杆菌睾丸免疫染色

摘要

探索更多视频

此视频中的章节

直立成像

用于活、完整组织中细胞分部分析的果蝇拉瓦尔和普帕尔睾试验的制备

全卵巢免疫荧光、清除和多光子显微镜用于小鼠发育中卵巢储备的定量3D分析

夹层和染色果蝇幼虫卵巢

果蝇蛹腹部免疫

在飞睾丸成像中心体

精子发生的细胞学分析：Live和固定制剂

后期阶段的胚胎和幼虫的超声，促进免疫荧光染色

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

亚细胞结构的超分辨率活细胞成像

用于大型精子细胞三维共焦分析的全山 嗜血杆菌 睾丸免疫染色

摘要

探索更多视频

此视频中的章节

直立成像

用于活、完整组织中细胞分部分析的果蝇拉瓦尔和普帕尔睾试验的制备

全卵巢免疫荧光、清除和多光子显微镜用于小鼠发育中卵巢储备的定量3D分析

夹层和染色果蝇幼虫卵巢

果蝇蛹腹部免疫

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