夹层和染色果蝇幼虫卵巢
Summary
壁龛和干细胞如何在发展过程中形成的，是一个具有实际意义的重要问题。在<em>果蝇</em>卵巢，生殖系干细胞和体龛的形式在幼虫发育。本视频演示了如何解剖，染色和安装从晚三龄（LL3）女性性腺<em>果蝇</em>幼虫。
Abstract
许多器官取决于其发展的干细胞，胚胎发育过程中和在成年生活中的保养或维修。因此，了解如何干细胞的形式，以及如何与环境互动的了解发展，动态平衡和疾病的关键。曾担任果蝇卵巢生殖系干细胞与他们的躯体支持细胞（利基）1，2（GSCs）的相互作用的一个有影响力的模型。的利基和GSCs已知的位置，再加上基因操纵他们的能力，使研究人员阐明了各种干细胞及其龛3-12之间的相互作用。
尽管控制GSC维护和分化机制的信息财富，相对鲜为人知的是，如何GSCs和他们的体龛在发展过程中形成。约18体龛，其细胞成分，包括终端长丝和帽细胞（图1），在第三幼虫龄 13-17形式。 GSCs源于原始生殖细胞（PGCs）。 PGCS增殖早期幼虫阶段，但形成的利基的PGCS分组成为GSCs 7，16，18，19。总之，体利基细胞的GSCs一个功能单位，生产鸡蛋整个生物体的寿命。
就形成的GSC单位许多问题仍然悬而未决。如壁龛和干细胞前体的前体细胞，或代内PGCS不对称，因为他们成为GSCs之间的协调过程，最好是能在幼虫研究。然而，有条不紊幼虫卵巢发育的研究是身体具有挑战性。首先，幼虫卵巢小。即使在后期幼体阶段，他们只有100μm的跨越。此外，卵巢是透明的，嵌入在白色脂肪组织。在这里，我们描述一个孤立晚三龄（LL3） 果蝇幼虫，用荧光抗体染色，卵巢的一步一步的协议。我们提供了一些问题，如定位卵巢，染色和洗涤不下沉的组织，确保抗体渗透到组织的技术解决方案。该协议可应用于早期幼虫期和幼虫睾丸以及。
Protocol
1。产卵 5天前清扫：允许交配女性躺在鸡蛋2-4个小时，用酵母补充的新鲜食品。为了得到同步和发达的幼虫，重要的是不要有人满为患培养（约30卵/25毫米瓶）。通常情况下，每小瓶7-16女性，取决于他们躺在。 2。选择幼虫准备9以及玻璃，夹层菜充满林格氏液（128毫米氯化钠，2MM KCL，1.8MM，4MM氯化钙氯化镁 2，35.5mM蔗糖，5MM HEPES pH值6.9） ?…
Discussion
本视频演示晚三龄幼虫卵巢的隔离和染色协议。要经常和可靠地执行这一协议，应注意以下几点：
- 同步和发达的幼虫，较拥挤，必须避免。
- 为了避免损失小的半透明卵巢，确保解剖脂肪体完好无损。这也将有助于在安装阶段的定位卵巢，尤其是当安装年轻（第二或第三龄开始）卵巢。
- 为了同时处理很多样品，使用细胞过滤器或其他的玩意儿。
- 确保永不干涸的样…
Declarações
The authors have nothing to disclose.
Acknowledgements
IM是由居里夫人重新整合的赠款支持。这项工作是由以色列科学基金资助没有的支持。 1146年至1108年，由海伦和马丁金梅尔在魏茨曼科学研究所的干细胞研究和Leir慈善基金会。
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| NaCl | J.T.Baker | |||
| Kcl | Merck | |||
| CaCl2 | Sigma-Aldrich | |||
| MgCl2 | Merck | |||
| Sucrose | J.T.Baker | |||
| Hepes | Sigma-Aldrich | |||
| PBS | Sigma-Aldrich | |||
| Triton X-100 | Sigma-Aldrich | |||
| Albumin Bovine Fraction V | MP Biomedicals | 160069 | ||
| Dumont biology tweezers 5 dumstar polished | FST Fine Science Tools | 11295-10 | ||
| Nickel plated pin holder | FST Fine Science Tools | 26018-17 | ||
| s.s minutien pins 0.1mm diam, 10mm long | FST Fine Science Tools | 26002-10 | ||
| 9 well plates 85X100 mm, 22mm o.d.x7mm deep | CORNING | 7220-85 | ||
| Stereo Microscope MZ 16.5 with a standard transmitted light base TL ST | Leica | |||
| 6 well plates | Costar | 3516 | ||
| Slides | Menzel Glaser GmbH | 798 | ||
| Cover slips | Corning | 2940-223 | ||
| Mounting media | Vectashield | H-1200 | ||
| Cell strainer | Falcon | FAL352350 | ||
| 1B1 antibody | Developmental Studies Hybridoma Bank | |||
| Anti-Traffic Jam | Laboratory of Dr. Dorothea Godt | |||
| Anti-Vasa | Laboratory of Dr. Ruth Lehmann | |||
| Anti β-Galactosidase | Cappel | |||
| Secondary Antibodies | Jackson ImmunoResearch |
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Tags
DissectionStainingDrosophila Larval OvariesStem CellsDevelopmentEmbryogenesisMaintenanceRepairAmbienteFruit FlyDrosophila MelanogasterGerm Line Stem Cells (GSCs)Somatic Support CellsNicheGenetic ManipulationInteractionsMechanismsDifferentiationSomatic NichesCellular ComponentsTerminal FilamentCap CellsLarval InstarPrimordial Germ Cells (PGCs)ProliferationFunctional UnitEggs
Citar este artigo
Maimon, I., Gilboa, L. Dissection and Staining of Drosophila Larval Ovaries. J. Vis. Exp. (51), e2537, doi:10.3791/2537 (2011).