这项研究得到了国家卫生研究院 (RO1 GM079351 到交流) 的支持。我们感谢 Godt 为她提供了有益的建议, 蛹卵巢解剖根据她的原始协议。我们还感谢艾米 Reilein 对手稿的帮助和评论。

1. EggLaying
<ol><li>结合大约十个男性和十五个女性成年果蝇的期望基因型的苍蝇在一瓶正常丰富的苍蝇食品补充酵母。为了避免瓶子过度拥挤, 允许交配雌性产卵不长于 2–4 h14。</li>
	<li>把这些成年人从瓶子里移到一个新瓶子里, 然后用小瓶子打开, 用苍蝇食物来对付不同的瓶子。允许卵在室温下发育成幼虫, 3–4天。</li>
</ol>2. 选择雌幼虫
<ol><li>使用柔软的毛刷湿润的细刷子, 做一个滚动运动与刷子沿着瓶子的墙上转移流浪的第三龄幼虫从瓶子到一个玻璃井填补了1x 磷酸盐缓冲盐水 (PBS)。把食物碎片从幼虫身上洗掉, 然后把它们转移到一个充满 1x PBS 的井里。</li>
	<li>用镊子将雄性和雌性幼虫分开。用一对大的, 圆形的, 和半透明的睾丸, 在身体的侧面, 大约2/3 从它的前端15下, 识别雄性幼虫。雌性幼虫平均比雄性大, 不透明, 而且有更小的性腺, 更难检测。</li>
	<li>选择抗雄性幼虫收集雌性幼虫。从井里收集至少十名女性。将雌幼虫放入一个单独的井中, 填充 1x PBS。</li>
	<li>用镊子将雌幼虫轻轻地转移到新的新鲜食物瓶中, 补充酵母。</li>
	<li>将小瓶与雌性幼虫放在一个黑暗的位置, 以方便 pupariation16。整个白天, 监测幼虫的 pupariation。当每个幼虫力场并且发展成一个预蛹, 圆预蛹反对小瓶并且记录近似时间, 当它第一次形成入预蛹。通过前气孔的突出和蛹壳形成的时机确定预蛹17。允许预蛹发展到所需的时间点 (以小时为单位, 蛹壳形成后, APF)。 注意: 任何在大约10小时间隔内蛹壳形成的动物都可以被认为是预蛹。蛹阶段开始约12小时后蛹壳形成后, 内部蜕皮已发生。</li>
</ol>3. 制备抗体染色蛹
<ol><li>
形成一个薄玻璃吸管, 用于清除蛹脂肪体的后期步骤。
	<ol>
<li>将巴斯德吸管的玻璃尖端融化在本生燃烧器上。当玻璃融化时, 用镊子将尖端从吸管的其他部分水平拉出, 形成更薄的尖端。</li>
		<li>冷却后, 折断吸管尖端的一小部分, 形成一个整洁的圆形开口。将灯泡连接到吸管的另一端。</li>
		<li>用 1x PBS 加载吸管。</li>
	</ol>
</li>
	<li>为了收割被粘在瓶子壁上的蛹, 在蛹和小瓶之间的接触区应用一滴水。在用湿润的细刷子将蛹从墙上轻轻地抬起之前, 先让蛋白胶溶解。将蛹转移到装有 1x PBS 的玻璃井中。为每个蛹奉献一个好的, 以避免过度拥挤的井。</li>
	<li>在抓蛹的后端与一对钳, 小心地撕裂前部的蛹的案件与另一对, 直到蛹的头是可见的。</li>
	<li>用镊子抓住蛹头最前端的尖端, 轻轻地将蛹从蛹的箱子中拉出。 注意: 脂肪体的一部分可能会在这个过程中溢出。</li>
	<li>将蛹的前半部分从后半部分分离并丢弃, 直到只有腹腔袋残留。</li>
	<li>
从腹腔袋中提取脂肪体细胞。
	<ol>
<li>一只手抓住腹部袋底部的井与镊子, 同时持有的薄玻璃吸管填充 1x PBS 在另一只手。</li>
		<li>瞄准袋子的开口的薄吸管尖和慢慢地吸管 1x PBS 入腹部洗掉周围的蛹卵巢的脂肪体细胞。 注意: 卵巢是一对小, 半透明, 纹状, 和长方形的结构, 应该留在腹部袋。</li>
		<li>洗去脂肪的身体细胞, 直到至少有2/3 的脂肪身体消失或直到卵巢可见附近的腹部袋开口。慢慢地执行这一步骤是非常重要的, 以免意外地将卵巢从腹部冲洗出来。</li>
	</ol>
</li>
	<li>检查油井, 检查卵巢是否在脂肪体细胞洗涤过程中溢出。如果卵巢在井内不可见, 他们应该留在腹部。如果卵巢出来了, 在井里放一个新的蛹, 重复这一步。</li>
	<li>将腹部转移到一个清晰的 coverglass 腔内, 内固定缓冲 (1x PBS, 4% 多聚甲醛), 并将盖子放在上面。为了确保足够数量的卵巢经得起染色和安装过程, 解剖更多的卵巢比需要。 注意: 固定缓冲液中含有毒性多聚甲醛。请佩戴适当的防护, 如手套, 安全眼镜,等。</li>
</ol>4. 免疫组化
<ol><li>在整个染色过程中, 使用镊子轻轻地将任何浮动卵巢向下推到盖玻璃的底部, 确保卵巢完全沉浸在抗体溶液中。</li>
	<li>将双面胶带放在 Nutator 的平坦表面上, 然后将腔 coverglass 放在粘合剂表面上。</li>
	<li>在室温下从步骤3.7 到15分钟, 在固定缓冲中孵化卵巢。</li>
	<li>冲洗卵巢三倍, 在 1x PBS 与1% 海卫 X-100 5, 10, 45 分钟 (总共1小时), 以允许彻底通透。</li>
	<li>将10% 正常山羊血清中的卵巢阻断在 1x PBS 中, 0.5% X-100 为30分钟。</li>
	<li>在600µL 的初级抗体选择稀释到适当的浓度在 1x PBS 与0.5% 海卫 X-100 4 摄氏度, 夜间孵化卵巢。</li>
	<li>三次冲洗卵巢5分钟 1x PBS 与0.5% 海卫 X-100。</li>
	<li>在600µL 的二次抗体中孵化卵巢2小时, 稀释到适当浓度的 1x PBS 中, 0.5% 的海卫 X-100 室温下。</li>
	<li>在 1x pbs 中冲洗卵巢两次, 5 分钟, 0.5% 海卫 X-100, 5 分钟, 1x pbs。</li>
</ol>5. 解剖和安装蛹卵巢
<ol><li>将一小滴 1x PBS 放到显微镜滑梯上。使用镊子将腹腔袋从腔 coverglass 转移到一个玻璃井填充 1x PBS。为每个蛹奉献一个好的, 以避免过度拥挤的井。</li>
	<li>用镊子将腹腔袋和任何剩余脂肪体撕成一裂。 注意: 卵巢, 这是一对小, 半透明, 纹状, 和长方形的结构, 应该在井内可见。卵巢通常被脂肪的身体紧紧包围, 所以确保所有脂肪体完全撕裂是很重要的。</li>
	<li>将卵巢从井中转移到显微镜滑梯上的 1x PBS 中, 方法是在钳的尖端之间抓住卵巢中心。这是非常重要的是使用一个牢固的抓地力, 而不挤压卵巢, 以免失去或摧毁他们在转移。当解决方案随着时间的推移而变干时, 添加几滴 1x PBS 到幻灯片中。</li>
	<li>一旦所有卵巢被解剖和转移到显微镜幻灯片, 吸管40µL 的安装介质到22毫米 x 22 毫米盖玻片, 并将盖玻片轻轻地放在卵巢的顶端。让幻灯片在成像前一夜干。</li>
</ol>

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作者没有要申报的利益冲突。

本议定书最关键和最困难的步骤是在固定前准备蛹卵巢。为了确保卵巢, 小和埋在蛹腹部的脂肪体细胞, 有足够的抗体, 这是重要的, 不仅撕裂一个大的开放, 在腹腔麻袋与钳, 但也提取脂肪体细胞, 阻碍卵巢的抗体。成功执行此步骤需要在巴斯德吸管灯泡上应用微妙的压力, 同时将脂肪体细胞从腹袋中清洗出来 (例如, 在这一步中, 在每秒的时间内都应留出腹部)。如果不使用温和的力量, 可能会...

干细胞研究使用果蝇卵巢已广泛扩展自第一个文件的干细胞利基1,2,3,4。随着沿袭追踪遗传工具的发展,果蝇卵巢解剖通常被用来研究干细胞的谱系和信号通路, 调节干细胞的维持、增殖和命运。有关这些信号通路的知识可能会对源于异常干细胞活动的癌症的潜在病因产生洞察力5,6,7。最近还显示, 在果蝇卵巢中, 称为卵泡干细胞 (开办) 的体细胞干细胞在其组织的许多方面都与哺乳动物的肠道干细胞有很大的相似性, 这些细胞有8。因此,果蝇卵巢是研究干细胞行为的一个非常有用的模型系统。
幼虫和成人卵巢为早期干细胞的发育和生态位的最终干细胞组织提供了线索, 蛹卵巢是生殖和体细胞重组和建立其身份的中间结构。9,10. 虽然有几项研究已经检查了蛹子房10、11、12、13的组织发展方面, 但关于分化和空间组织的问题依然存在。蛹发育期间的卵巢细胞类型。特别是, 开办的规格在这个期间发生。本协议概述了在所需的时间点解剖和染色蛹卵巢的方法, 这一技术可用于从幼虫到成年期详细分析蛹卵巢发育的时间过程实验。
为了解释蛹腹中蛹卵巢的体积小、半透明和难以接近的问题, 本协议利用诸如自定义的薄尖巴斯德吸管等工具去除腹部脂肪体组织阻碍抗体进入卵巢.在抗体染色过程中使用的一个清晰的腔内 coverglass, 可以使蛹的可见度更高, 并且可以在 "Nutator" 上摇动卵巢的温和平台。基于 Maimon 和基利波山14的幼虫卵巢解剖协议, 在染色过程的最初步骤中采用了相对较高浓度的海卫 X-100, 以最大限度地提高细胞膜通透和抗体进入卵巢细胞。

<table><tbody><tr><td>Dumont #5 Forceps, biology</td><td>Fine Scientific Tools</td><td>11252-20</td><td></td></tr><tr><td>Nunc Lab-Tek Chambered Coverglass</td><td>Thermo Fisher Scientific</td><td>155383</td><td></td></tr><tr><td>Dissection microscope</td><td>Nikon</td><td>SMZ-10A</td><td></td></tr><tr><td>Confocal Microscope</td><td>Carl Zeiss</td><td>LSM 700</td><td></td></tr><tr><td>Analysis software</td><td>Carl Zeiss</td><td>Zen</td><td></td></tr><tr><td>9 Depression Glass Spot Plates</td><td>Pyrex</td><td>7220-85</td><td></td></tr><tr><td>Pasteur pipet</td><td>Fisher Scientific</td><td>13-678-6B</td><td></td></tr><tr><td>Pasteur pipet bulb</td><td>Various vendors</td><td></td><td></td></tr><tr><td>Bunsen burner</td><td>Various vendors</td><td></td><td></td></tr><tr><td>Fisherfinest Premium Frosted Microscope Slides</td><td>Thermo Fisher Scientific</td><td>12-544-2</td><td></td></tr><tr><td>22 x 22 mm glass coverslips No 1</td><td>VWR</td><td>48366-067</td><td></td></tr><tr><td>Dapi Fluoromount-G</td><td>SouthernBiotech</td><td>0100-20</td><td></td></tr><tr><td>Double-sided tape</td><td>Scotch</td><td></td><td></td></tr><tr><td>Nutator</td><td>Clay Adams</td><td></td><td></td></tr><tr><td>Fine brush #0, #3-#5</td><td>Various vendors</td><td></td><td></td></tr><tr><td>Gilson Pipetman Starter Kit</td><td>Thomas Scientific</td><td>F167300</td><td>Contains p20, p200, p1000 pipettors</td></tr><tr><td>16% Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Dilute to 4% paraformaldehyde in 1x PBS</td></tr><tr><td>Triton</td><td>Sigma-Aldrich</td><td>9002-93-1</td><td></td></tr><tr><td>10x PBS</td><td>Ambion</td><td>AM9624</td><td>Dilute to 1x PBS</td></tr><tr><td>Normal Goat Serum</td><td>Jackson ImmunoResearch</td><td>5000121</td><td>Dilute to 10% normal goat serum in PBST with 0.5% Triton concentration</td></tr><tr><td>Primary antibodies (in protocol: 7G10 anti-Fasciclin III diluted 1:250, rabbit anti-phosphohistone H3 diluted 1:1000)</td><td>Various vendors (in protocol: Developmental Studies Hybridoma Bank, Millipore)</td><td></td><td>Dilute in PBST with 0.5% Triton concentration</td></tr><tr><td>Secondary antibodies (in protocol: Alexa-546, FITC-conjugated anti-rabbit serum)</td><td>Various vendors (in protocol: Molecular Probes, Jackson ImmunoResearch Laboratories, Inc.)</td><td></td><td>Dilute in PBST with 0.5% Triton concentration</td></tr><tr><td>Fly vials</td><td>Denville Scientific</td><td>V9406</td><td></td></tr><tr><td>Cotton Balls, For Wide Vials</td><td>Genesee Scientific</td><td>51-102W</td><td></td></tr><tr><td>Yeast, Bakers Dried Active</td><td>MP Biomedicals</td><td>101400</td><td></td></tr><tr><td>Fly food</td><td>Produced in laboratory</td><td></td><td>Mixture of water, brewer&#039;s yeast, cornmeal, molasses, agar, EtOH, penicillin, methyl 4-hydrobenzoate, and propionic acid</td></tr><tr><td>Male and female &lt;em&gt;Drosophila&lt;/em&gt; flies (genotype used in protocol: yw; P[Fz3-RFP, w+]/TM2)</td><td>Bloomington Drosophila Stock Center</td><td></td><td></td></tr></tbody></table>

dissection and staining of drosophila pupal ovaries

与成年的果蝇卵巢不同, 蛹卵巢由于其体积小、半透明和在蛹的情况下被包裹而难以进入和检查。解剖蛹卵巢的挑战还在于它们在蛹中的物理位置: 卵巢被蛹腹部内的脂肪体细胞所包围, 必须除去这些脂肪细胞, 以允许适当的抗体染色。为了克服这些挑战, 本协议利用自定义的巴斯德滴管从蛹腹部提取脂肪体细胞。此外, 在染色过程中使用腔内 coverglass 代替离心管, 以提高蛹的可见度。然而, 尽管本议定书使用的工具有这些和其他优点, 但由于蛹卵巢体积小, 成功地执行这些技术可能还需要几天的实践。本议定书所概述的技术可应用于蛹发育不同阶段的卵巢分析的时间过程实验。

成功执行此过程应导致清晰的抗体染色, 揭示了一个果蝇蛹卵巢的结构和细胞组织。免疫组织化学在本议定书中概述可用于识别常见的细胞类型在幼虫和成人卵巢染色。从群单元派生的蛹茎细胞18 (由白色 Fasciclin III 所概述) 显示在图 3中。除了突出蛹卵巢的细胞组织外, 特定于细胞增殖的抗体染色 (如以绿色显示的<strong class="xf...

Watch this Scientific Journal Video about Dissection and Staining of Drosophila Pupal Ovaries at JoVE.com

Dissection and Staining of Drosophila Pupal Ovaries

果蝇卵巢是研究干细胞小生境发育的优良模型系统。虽然已经公布了解剖幼虫和成人卵巢的方法, 蛹卵巢解剖需要不同的技术, 没有详细发表。在这里, 我们概述了解剖, 染色和安装蛹卵巢的协议。

果蝇蛹卵巢的解剖与染色

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

dissection-and-staining-of-drosophila-pupal-ovaries

Research

JoVE Journal

Developmental Biology

Dissection and Staining of Drosophila Pupal Ovaries

9.3K 次观看.  Columbia University. 果蝇卵巢是研究干细胞小生境发育的优良模型系统。虽然已经公布了解剖幼虫和成人卵巢的方法, 蛹卵巢解剖需要不同的技术, 没有详细发表。在这里, 我们概述了解剖, 染色和安装蛹卵巢的协议。

视频: Dissection and Staining of Drosophila Pupal Ovaries

Dissection of Drosophila Ovaries

剖析果蝇卵巢

科研

生物学

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 ...

发育生物学

Visualizing the Effects of Oxidative Damage on Drosophila Egg Chambers using Live Imaging

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ribonucleoprotein particle movement, mRNA localization, organelle movement, and cytoskeletal dynamics. There are several methods for live imaging that have been developed. Due to the fact that each method involves dissecting individual ovarioles placed in media or halocarbon oil, cellular damage due to hypoxia and/or physical manipulation will inevitably occur over time. One downstream effect of hypoxia is to increase oxidative damage in the cells. The purpose of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries after induction of controlled cellular damage. Here, we use hydrogen peroxide to induce cellular oxidative damage and give examples of the effects of such damage on two subcellular structures, mitochondria and Clu bliss particles. However, this method is applicable to any subcellular structure. The limitations are that hydrogen peroxide can only be added to aqueous media and would not work for imaging that uses halocarbon oil. The advantages are that hydrogen peroxide is readily available and inexpensive, acts quickly, its concentrations can be modulated, and oxidative damage is a good approximation of damage caused by hypoxia as well as general tissue damage due to manipulation.

The objective of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries.

使用实时成像可视化氧化损伤对果蝇卵室的影响

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ...

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment and maintenance of epithelial tissue morphology and organ morphogenesis. Moreover, the BM is essential for tissue modeling, serving as a signaling platform, and providing external forces to shape tissues and organs. Despite the many important roles that the BM plays during normal development and pathological conditions, the biological pathways controlling the intracellular trafficking of BM-containing vesicles and how basal secretion leads to the polarized deposition of BM proteins are poorly understood. The follicular epithelium of the Drosophila ovary is an excellent model system to study the basal deposition of BM membrane proteins, as it produces and secretes all major components of the BM. Confocal and super-resolution imaging combined with image processing in fixed tissues allows for the identification and characterization of cellular factors specifically involved in the intracellular trafficking and deposition of BM proteins. This article presents a detailed protocol for staining and imaging BM-containing vesicles and deposited BM using endogenously tagged proteins in the follicular epithelium of the Drosophila ovary. This protocol can be applied to address both qualitative and quantitative questions and it was developed to accommodate high-throughput screening, allowing for the rapid and efficient identification of factors involved in the polarized intracellular trafficking and secretion of vesicles during epithelial tissue development.

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane is essential for tissue and organ morphogenesis during development. To better understand the mechanisms leading to proper placement of this structure, the protocol presented describes methods to visualize and characterize the intracellular trafficking and secretion of basement membrane proteins in epithelial cells using confocal and super-resolution microscopy.

果蝇卵生过程中极化细胞内运输和基底膜蛋白分泌的共聚焦和超分辨率成像

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment ...

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 ...

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 ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

解剖和免疫染色成虫盘的<em&gt;果蝇</em

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

研究线粒体结构与功能<em&gt;果蝇</em&gt;卵巢

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and diseases, such as obesity and fatty liver. Usually, lipids are stored in lipid droplets, which are the multifunctional lipid storage organelles in cells. Lipid droplets vary in size and number in different tissues and under different conditions. It has been reported that lipid droplets are tightly controlled through regulation of its biogenesis and degradation. In Drosophila melanogaster, the oenocyte is an important tissue for lipid metabolism and has been recently identified as a human liver analogue regarding lipid mobilization in response to stress. However, the mechanisms underlying the regulation of lipid droplet metabolism in oenocytes remain elusive. To solve this problem, it is of utmost importance to develop a reliable and sensitive method to directly visualize lipid droplet dynamic changes in oenocytes during development and under stressful conditions. Taking advantage of the lipophilic BODIPY 493/503, a lipid droplet-specific fluorescent dye, described here is a detailed protocol for the dissection and subsequent lipid droplet staining in the oenocytes of Drosophila larvae in response to starvation. This allows for qualitative analysis of lipid droplet dynamics under various conditions by confocal microscopy. Furthermore, this rapid and highly reproducible method can also be used in genetic screens for indentifying novel genetic factors involving lipid droplet metabolism in oenocytes and other tissues.

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Presented here are detailed methods for the dissection and lipid droplet staining of oenocytes in Drosophila larvae using BODIPY 493/503, a lipid droplet-specific fluorescent dye.

果蝇幼虫的欧诺细胞的解剖和脂滴染色

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and ...

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent adult integument. Their immobility and transparency make them ideal for in vivo live imaging experiments. Many studies have focused on the dorsal epithelial monolayer of the pupal&#160;notum because of its accessibility and relatively large size. In addition to the studies of epithelial mechanics and development, the notum has been an ideal tissue to study wound healing. After an injury, the entire epithelial repair process can be captured by live imaging over 6-12 h. Despite the popularity of the notum for live imaging, very few published studies have utilized fixed notum samples. Fixation and staining are common approaches for nearly all other Drosophila tissues, taking advantage of the large repertoire of simple cellular stains and antibodies. However, the pupal notum is fragile and prone to curling and distortion after removal from the body, making it challenging to complement live imaging. This protocol offers a straightforward method for fixing and staining the pupal notum, both intact and after laser-wounding. With this technique, the ventral side of the pupa is glued down to a coverslip to immobilize the pupa, and the notum is carefully removed, fixed, and stained. The notum epithelium is mounted on a slide or between two coverslips to facilitate imaging from the tissue&#39;s dorsal or ventral side.

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The present protocol details the preparation and visualization of fixed tissue of the Drosophila pupal notum. It can be used for either intact or wounded tissue, and the original architecture of the tissue is preserved. The procedures for dissecting, fixing, and staining are all described in this article.

解剖、固定和可视化 果蝇 蛹

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent ...

果蝇蛹卵巢的解剖与染色

摘要

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剖析果蝇卵巢

直立成像

使用实时成像可视化氧化损伤对果蝇卵室的影响

果蝇卵生过程中极化细胞内运输和基底膜蛋白分泌的共聚焦和超分辨率成像

夹层和染色果蝇幼虫卵巢

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解剖和免疫染色成虫盘的

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解剖、固定和可视化果蝇蛹

果蝇蛹卵巢的解剖与染色

摘要

探索更多视频

此视频中的章节

剖析果蝇卵巢

直立成像

使用实时成像可视化氧化损伤对果蝇卵室的影响

果蝇卵生过程中极化细胞内运输和基底膜蛋白分泌的共聚焦和超分辨率成像

夹层和染色果蝇幼虫卵巢

果蝇蛹腹部免疫

解剖和免疫染色成虫盘的

研究线粒体结构与功能

果蝇幼虫的欧诺细胞的解剖和脂滴染色

解剖、固定和可视化 果蝇 蛹

解剖、固定和可视化果蝇蛹