果蝇蛹卵巢的解剖与染色
Summary
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。
Abstract
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。
Introduction
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。
Protocol
1. EggLaying 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。 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。 2. 选择雌幼虫 使用柔软…
Representative Results
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…
Discussion
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…
Declarações
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Dumont #5 Forceps, biology | Fine Scientific Tools | 11252-20 | |
| Nunc Lab-Tek Chambered Coverglass | Thermo Fisher Scientific | 155383 | |
| Dissection microscope | Nikon | SMZ-10A | |
| Confocal Microscope | Carl Zeiss | LSM 700 | |
| Analysis software | Carl Zeiss | Zen | |
| 9 Depression Glass Spot Plates | Pyrex | 7220-85 | |
| Pasteur pipet | Fisher Scientific | 13-678-6B | |
| Pasteur pipet bulb | Various vendors | ||
| Bunsen burner | Various vendors | ||
| Fisherfinest Premium Frosted Microscope Slides | Thermo Fisher Scientific | 12-544-2 | |
| 22 x 22 mm glass coverslips No 1 | VWR | 48366-067 | |
| Dapi Fluoromount-G | SouthernBiotech | 0100-20 | |
| Double-sided tape | Scotch | ||
| Nutator | Clay Adams | ||
| Fine brush #0, #3-#5 | Various vendors | ||
| Gilson Pipetman Starter Kit | Thomas Scientific | F167300 | Contains p20, p200, p1000 pipettors |
| 16% Paraformaldehyde | Electron Microscopy Sciences | 15710 | Dilute to 4% paraformaldehyde in 1x PBS |
| Triton | Sigma-Aldrich | 9002-93-1 | |
| 10x PBS | Ambion | AM9624 | Dilute to 1x PBS |
| Normal Goat Serum | Jackson ImmunoResearch | 5000121 | Dilute to 10% normal goat serum in PBST with 0.5% Triton concentration |
| Primary antibodies (in protocol: 7G10 anti-Fasciclin III diluted 1:250, rabbit anti-phosphohistone H3 diluted 1:1000) | Various vendors (in protocol: Developmental Studies Hybridoma Bank, Millipore) | Dilute in PBST with 0.5% Triton concentration | |
| Secondary antibodies (in protocol: Alexa-546, FITC-conjugated anti-rabbit serum) | Various vendors (in protocol: Molecular Probes, Jackson ImmunoResearch Laboratories, Inc.) | Dilute in PBST with 0.5% Triton concentration | |
| Fly vials | Denville Scientific | V9406 | |
| Cotton Balls, For Wide Vials | Genesee Scientific | 51-102W | |
| Yeast, Bakers Dried Active | MP Biomedicals | 101400 | |
| Fly food | Produced in laboratory | Mixture of water, brewer's yeast, cornmeal, molasses, agar, EtOH, penicillin, methyl 4-hydrobenzoate, and propionic acid | |
| Male and female Drosophila flies (genotype used in protocol: yw; P[Fz3-RFP, w+]/TM2) | Bloomington Drosophila Stock Center |
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Citar este artigo
Park, K. S., Godt, D., Kalderon, D. Dissection and Staining of Drosophila Pupal Ovaries. J. Vis. Exp. (133), e56779, doi:10.3791/56779 (2018).