现场共聚焦显微镜对斑马鱼的胚胎脑成像

Biology

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Summary

在这段视频中,我们表现出的方​​法,通过它来分析生活在单细胞共聚焦显微镜分辨率的斑马鱼胚胎发育中的脊椎动物大脑。这包括我们注入单细胞的斑马鱼胚胎的方法,通过它,随后装载和图像的大脑发育。

Cite this Article

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Graeden, E., Sive, H. Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy. J. Vis. Exp. (26), e1217, doi:10.3791/1217 (2009).

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Abstract

在这段视频中,我们展示了我们的实验室已发展到分析细胞形态的变化和需要弯曲和折叠的发展斑马鱼脑(Gutzman等,2008)的重排的方法。这种分析提供了一次脊椎动物大脑的三维结构的发展所需的基本细胞生物学的新认识,并显着提高我们的能力,以研究神经管的形态发生。胚胎斑马鱼的大脑形脑室神经上皮内膨胀的开端,在18小时后受精(HPF)。 24 HPF,神经管的形态发生的最初步骤,是完整的。使用这里介绍的方法,在细胞阶段的胚胎基因编码的膜,有针对性的绿色荧光蛋白(memGFP)注射。后注射和孵化,胚胎,目前18至24 HPF,安装,倒置,在琼脂糖和激光共聚焦显微镜成像。值得注意的是,斑马鱼的胚胎是透明的,使其成为理想的荧光成像系统。虽然我们的分析集中在中脑,后脑边界和后脑,这种方法可以延长任何地区的分析,在深度为80-100微米的斑马鱼。

Protocol

1。准备注射基因

  1. 在此过程中使用的mRNA是由质粒编码CAAX - EGFP(memGFP)mRNA的转录。首先线性化的质粒,根据2008年等,Gutzman。
  2. 然后抄写memGFP mRNA的使用mMessage mMachine套件。
  3. 稀1μg/μl,导致mRNA的等份,并储存在-80 ° C。
  4. 在一个细胞阶段的胚胎车道​​的宽度,准备1%琼脂糖的注塑模具。
  5. 在注射前一天,成立了分离的女性,男性的交配笼。
  6. 注射日,拉毛细管,使用微量车夫准备注射针。

2。注射的mRNA

  1. 在注射日,解冻1μg/μlmemGFP的mRNA在冰上的等份。
  2. 在水稀释的mRNA 1:5的200ng/μl最终浓度,并保持在冰上。
  3. 负载200ng/μl准备memGFP表达加入1μl毛细管针。
  4. 装入一个天然气为动力的微量的一个显微针。
  5. 调整的注射量1nl。
  6. 野生鱼交配笼前一天拉的分隔。
  7. 收集胚胎,尽快为他们规定。东方他们就在身边离的显微单细胞胚胎培养基(韦斯特,1995年)所覆盖的琼脂糖注塑模具。
  8. 注入通过蛋壳和蛋黄等,直接存入单细胞的mRNA。注入约50%的胚胎实验。不要注入的细胞分为。
  9. 28 ° C过夜胚胎培养基孵育胚胎

3。安装和成像胚胎

  1. 挂载和形象的胚胎,删除感兴趣的时间点之前的钳体视显微镜下一个一小时的胚胎绒毛膜。
  2. 准备安装多达4胚胎幻灯片。
    • 使用有机硅真空润滑脂密封盖玻片上的一个特别设计的一个洞,大约直径1cm 2mm厚的塑料滑动的底面。
    • 0.7%琼​​脂糖在幻灯片的填充孔,静置约20分钟或直到硬化
    • 一旦琼脂糖凝固后,取出一个小插件,使用200μL移液器形成圆柱形的孔,在安装每个胚胎。
  3. 广场上的琼脂糖凝胶填充的幻灯片,在解剖显微镜下的胚胎。添加tricaine50μL(韦斯特,1995年)麻醉胚胎。
  4. 使用镊子对基础盖玻片利益与大脑或地区在琼脂糖圆柱孔定位的胚胎。
  5. 硅真空润滑脂保护另一盖玻片盖在琼脂糖胚胎。
  6. 图片胚胎使用一个倒置,荧光,激光扫描或旋转盘共聚焦显微镜。在63X或更高收集单个细胞内的神经上皮的高清晰度图像的图像。
  7. 图像TIFF文件从LSM软件出口和使用Photoshop进行分析。

4。代表性的成果/成果

在图1所示是24 HPF斑马鱼神经上皮中脑和后脑心室之间有代表性的共聚焦图像。每个单元是与膜结合的GFP标记。


图1。现场共焦成像一个memGFP 24 HPF注入斑马鱼的胚胎。
(一)神经上皮与GFP的概述的每个细胞的胚胎24 HPF。成像区分开的中脑和后脑脑室(M,H),形成了中脑,后脑边界。

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Discussion

在这段视频中,我们表现出的mRNA注射到单细胞的斑马鱼胚胎的方法。在这里,我们使用一种膜定位GFP标记每个细胞的基因编码。然后,我们演示了如何安装和形象在单细胞分辨率的发育中的大脑。这项技术使我们能够研究一种新型的细胞形态变化,基部收缩,形成中脑,后脑边界(Gutzman等,2008)所需。类似的分析等现象有可能显着扩大我们对脊椎动物的形态发生和底层细胞生物学的理解。

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Acknowledgements

谁最先开发这项技术在Sive实验室的博士詹妮弗Gutzman许多感谢。达纳 - 法伯癌症研究所的波士​​顿,马萨诸塞,慷慨地提供CAAX - GFP基因的质粒编码也感谢JB绿色。白石WM凯克基金会生物成像设施进行共焦成像。 HS是由NIHMH70926支持。 EG是支持由美国国家科学基金会博士后奖学金。

Materials

Name Type Company Catalog Number Comments
SeaKem GTG agarose Reagent Lonza Inc. 50027
3-aminobenzoic acid ethyl ester (Tricaine) Reagent Sigma-Aldrich A-5040
Capillary tubing Tool Frederick Haer and Co. 30-30-1 Borosil 1.0mmOD x 0.5mm ID/fiber 100mm
Silicone vacuum grease Reagent VWR international W0S717
Forceps Tool Fine Science Tools 11232-20 Dumont #5 Bio Inox
1-200 μl Pipette tips Tool USA Scientific, Inc. 111-0806 These are the correct size for 24 hpf embryos.
mMessage mMachine transcription kit Reagent Ambion AM1340 SP6 RNA polymerase
Zeiss LSM 510 scanning confocal Microscope Carl Zeiss, Inc.
Micromanipulator Tool Narishige International
Microinjector Tool Harvard Apparatus PLI-100
Micropipette puller Tool Sutter Instrument Co.

DOWNLOAD MATERIALS LIST

References

  1. Gutzman, J. H., Graeden, E., Sive, H. Formation of the zebrafish midbrain-hindbrain boundary constriction requires laminin-dependent basal constriction. Mech Dev. 125, (974), 11-12 (2008).
  2. Westerfield, M. The Zebrafish Book: a Guide for the Laboratory Use of Zebrafish. University of Oregon Press. Eugene, Oregon. (1995).

Comments

7 Comments

  1. Dear doctor: I think the method you development is very useful and easy to follow in our lab. However, I found that you use a specially designed ²mm-thick plastic slide with a hole approximately 1cm in diameter. Could you offer me the information about the company you bought this slide from? Or is this slide just a custom-made product? For I don't find the proper slide in our place. Looking forward for your reply. Best Regards, Li Ting

    Reply
    Posted by: Anonymous
    April 14, 2009 - 6:55 AM
  2. Li Ting, The plastic slides we use are custom made here at the machine shop at MIT.  The dimensions are the same as a regular microscope slide, but with a ² mm thickness and a single hole in the center that is approximately 1 cm in diameter.

    Reply
    Posted by: Anonymous
    April 15, 2009 - 8:44 AM
  3. Li Ting, The plastic slides we use are custom made here at the machine shop at MIT.  The dimensions are the same as a regular microscope slide, but with a ² mm thickness and a single hole in the center that is approximately 1 cm in diameter. Good luck, Jen Gutzman  

    Reply
    Posted by: Anonymous
    April 15, 2009 - 8:48 AM
  4. Li Ting,   As suggested by Dr. Jen Gutzman, also in our lab, our slides were custom-made by the MIT machine shop. The dimensions are the same as a regular microscope slide, but with a ² mm thickness and a single hole in the center that is approximately 1 cm in diameter. If you are having trouble getting them made, please contact me directly. We may be able to set up an order system through the machine shop here.   Best, Ellie Graeden

    Reply
    Posted by: Anonymous
    April 30, 2009 - 12:41 PM
  5. Dear
    Ellie Graeden & Hazel Sive
    First of all thanks for a nice publication entitled " Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy". This publication is very useful for me as I am also working with zebrafish and currently I am pursuing my PhD at Center for Clinical Research in Neuropsychiatry,Graylands Hospital & Center of Excellence for Alzheimer's Disease Research & Care, Perth, Australia. I will be highly obliged if you could send me the full text of your publication. Do your university provide a short term training for some techniques related to Zebrafish Research, if yes please let me know, I am highly interested. I shall be thankful to you for your kind help
    Cheers
    Avdesh

    Reply
    Posted by: Anonymous
    September 22, 2009 - 8:22 PM
  6. Dear Dr. Sive,
    Could yuo please send me a your paper entitled " Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy". In our imaging lab, we have initiated establishing zebrafish models for oncology. Your publication wil be a very helpful for us. I will be thankful if you could send me your above-mentioned publication and otehr related publications too.
    With best regards,
    Nishigandha Naik

    Reply
    Posted by: Anonymous
    July 5, 2011 - 6:58 AM
  7. What is the little loop tool you're using to align the embryos? It looks like it might be easier to use than the tool I currently use. Is it just a blackhead remover?

    Reply
    Posted by: Brandon B.
    May 29, 2012 - 1:31 AM

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