斑马鱼胚胎眼组织显微切割的
Summary
本文介绍的方法microdissect与视网膜色素上皮细胞附着，从一到三天受精胚胎斑马鱼视网膜。
Abstract
斑马鱼眼睛发育的模式，因为其快速流行的动物进行研究<em>前宫内</em>发展和良好的繁殖力。受精后3天（DPF），幼虫会显示第一视觉反应。许多基因已被确定为控制适当的眼球发育，但我们正在从一个潜在的遗传结构的完整的理解远。全基因组基因表达分析是一个有用的工具，以澄清对眼睛发育的基因调控网络。然而，在斑马鱼的胚胎眼的小尺寸使得它具有挑战性的，以取得完整和纯粹的眼部组织表达分析。例如，2和第3天之间的眼睛前后的长度仅约为200-300微米，而镜头的直径为100微米少。此外，基本视网膜视网膜色素上皮（RPE）仅仅是一个单层上皮。虽然可以从整个胚胎中获得的基因的表达谱，他们不准确地代表这些组织中的表达。因此，纯粹的组织必须得到一个成功的眼睛发育的基因表达图谱。为了解决这个问题，我们已开发出一种方法来microdissect附1-3 DPF与视网膜色素上皮完整的视网膜和视网膜，其中包括眼的形态发生的主要阶段。所有的程序可以做到的标准立体显微镜下用细镊子和一般实验室用品。视网膜剥离，单层RPE细胞被删除，去皮刷牙行动和培养板剥离面的视网膜色素上皮残余的优惠坚持。对于视网膜色素上皮附加视网膜剥离，视网膜色素上皮坚持夹层板被删除之前，剥离，使RPE细胞可以与视网膜完全保留。一个小心解除这个组织的行动能够有效地分离推定脉络膜和巩膜。镜头可以在这两种情况下，由化学蚀刻钨针删除。总之，我们的方法可以获取完整的眼部组织，并已成功地利用研究斑马鱼视网膜组织特异性表达谱<sup> 1，2</sup>和视网膜色素上皮细胞<sup> 3</sup>。
Protocol
Discussion
斑马鱼眼组织显微切割可以有效地获得完整的视网膜和视网膜色素上皮附加视网膜。这大大助攻表达的研究，涉及到一个特定的眼组织（即视网膜色素上皮）。事实上，我们已经成功地利用这些程序获得整个视网膜和视网膜色素上皮 3 RNA的表达谱。这些配置文件的实用工具，是我们最近的途径和基因家族在视网膜分化突变2扰动识别，大力支持。在视网膜的解剖程序的最关键步骤?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作是由一个在美国普渡大学生物科学系的启动资金支持。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Cordless pestle motor | VWR | 47747-370 | ||
| DC power supply | Lascar | PSU130 | Any DC supply would work. The specific voltage of a different machine will need further optimization. | |
| Disposable pestle & microtube, 1.5 mL (DNase, RNase and pyrogen-free) | VWR | 47747-366 | These are used for tissue collection in TRIzol for expression analysis. | |
| Dumont #5 forceps, Tips: 0.05 x 0.01mm, Inox | World Precision Instruments | 500341 | Fine tip dimension is desirable but is not inflexible, as one may need to sharpen the tip from time to time. | |
| Dumont #5SF forceps, Tips: 0.025 x 0.005mm, Inox | Fine Science Tools | 11252-00 | Fine tip dimension is desirable but is not inflexible, as one may need to sharpen the tip from time to time. | |
| Falcon polystyrene culture plates, 60 X 15 mm | BD Biosciences | 351007 | These plates are used as dissection plates. | |
| Olympus SZX16 Stereomicroscope | Olympus | SZX16 | Any stereomicroscope would work. We used Leica stereomicroscope in previous studies1-3 without any issues. We also use the 1X objective exclusively for the dissection even though we have a 2X objective installed. | |
| Sharpening stone | Fine Science Tools | 29008-01 | Use this to sharpen the tip of the forceps if necessary | |
| Thermo plate | Tokai Hit | MATS-U55SZX2B | This is used to maintain the temperature of the tissue throughout dissection and minimize the influence of temperature fluctuation on gene expression. We also put the whole microscope in an environmentally controlled room at 28°C during dissection in previous studies1-3 with good success. | |
| Trizol, 100 mL | Invitrogen | 15596-026 | ||
| tungsten wire, 0.015 inch diameter | World Precision Instruments | TGW1510 | ||
| Wooden Applicator | Puritan | 807 | This is used for holding the chemically-etched tungsten needle. |
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