果蝇幼虫NMJ夹层

Biology

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Summary

此协议示范如何解剖

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Brent, J. R., Werner, K. M., McCabe, B. D. Drosophila Larval NMJ Dissection. J. Vis. Exp. (24), e1107, doi:10.3791/1107 (2009).

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Abstract

Protocol

在您开始之前

  1. 所有的文化应该是生长在25 ° C
  2. HL3.1清扫缓冲区可能提前做好准备。以50毫升的HL3.1等份,并保持它在冰上。
  3. 准备在HL3.1 15毫升新鲜的3.5%的甲醛。保持它在冰上。

幼虫解剖

  1. 放一个寒冷HL3.1的夹层板的下降。这将保持干燥和眩晕的动物,使其更容易与动物。
  2. 长镊子 ,选择一个流浪的第三龄幼虫和HL3.1下降。
  3. 首先,使用短钳把握minutien针 。放置后气孔之间的引脚。动物通常会试图抓取从引脚本身拉伸,使它更容易为您放置在头口附近挂钩的幼虫的一个引脚正视。纵向伸展的动物。这将有助于您最大限度地就可以实现在切割过程中暴露的体壁量。注:如果您选择引脚头,动物可能会引脚环绕其身,或使其难以引脚,它的身体上来回鞭。通常你可以反删除HL3.1,并再次把一个新鲜的冷下降惊人的动物。灵巧的科学家也可以只抓动物和举行到位。
  4. 使用弹簧剪刀水平切口,只是前幼虫背侧后针。切口放入一个刀片剪刀,沿背中线对幼虫的喙的垂直切割。在动物的讲台,使针左侧和右侧横切口。注:完成的结果看起来应该像对我幼虫背侧沿rostrocaudal轴。
  5. 取出的器官。开始把一些额外的下降HL3.1幼虫。这将使机关浮出来的身体,让你更容易将其删除。首先,取出气管系统。二,使用镊子抢其余的器官,并删除它们。
  6. 在第3步,左瓣体壁和右瓣。引脚以顺时针为了确保横向和纵向拉伸体壁的襟翼。

固定

修正在3.5%甲醛25分钟在HL3.1,每个动物。洗两次HL3.1动物为5分​​钟。

存储

删除引脚和幼虫转移到1.5 ml管中含有1 × PBS。如果您计划NMJ使用融合荧光标记的图像,你应该在两天内的动物形象。您可能会存储一个星期的解剖幼虫在4 ° C

代表性的成果

有几个关键组件的正确解剖果蝇幼虫。首先,必须格外小心,不损伤肌肉,特别是肌肉4,6,和7。这些是最​​流行的肌肉研究,如果它们已损坏丢弃圆角的准备和解剖其他动物。其次,必须确保该动物是最大限度地伸展,使每一块肌肉和NMJ可以区分。

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Discussion

在此视频中演示的剥离技术可用于准备各种实验技术的果蝇幼虫。如果荧光蛋白标记,幼虫可安装和成像立即。否则,免疫,可为了纪念特定的突触车厢。此外,电可以轻松地进行解剖幼虫,以评估神经递质的运作。

果蝇 NMJ年初以来,照亮它的基本结构和功能的研究已取得大受欢迎。1-4许多果蝇NMJ的发展的研究中已确定的分子脊椎动物保守。 4因此,许多通过果蝇 NMJ的研究中吸取的见解,是适用于一切众生突触生物学。一些可能的应用包括参与轴突导向分子的研究,运动神经元疾病,学习和记忆。

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Materials

Name Company Catalog Number Comments
Stereomicroscope “Stemi” 2000 Carl Zeiss, Inc. 495101-9804-000
Light Source KL 1500 LCD Carl Zeiss, Inc. 000000-1063-181
3mm Vannas Spring Scissors Fine Science Tools 15000-0
Dumont SS Forceps Fine Science Tools 11200-33 Long forceps
Dumont #5 Forceps Fine Science Tools 11252-20 Short forceps
SylGard 182 Silicone Elastomer Kit Dow Corning 3097358-1004 Used to make dissection plates
Formaldehyde Sigma-Aldrich 252549-25ML
Stainless Steel Minutien Pins -0.1 MM Diameter Fine Science Tools 26002-10
HL3 Dissection Buffer: (in mM) 70 NaCl, 5 KCl, 0.2 CaCl2, 20 MgCl2, 10 NaHCO3, 5 trehalose, 115 sucrose, and 5 HEPES, pH 7.3.

Sylgard Dissecting Plate: Mix gently (to avoid bubbles) 10:1in a beaker. Pour mixture into Petri dish (any size). Allow SylGard to cure overnight in 37C incubator.


Sylgard Dissecting Plate: Mix gently (to avoid bubbles) 10:1in a beaker. Pour mixture into Petri dish (any size). Allow SylGard to cure overnight in 37C incubator.

Sylgard Dissecting Plate: Mix gently (to avoid bubbles) 10:1in a beaker. Pour mixture into Petri dish (any size). Allow SylGard to cure overnight in 37C incubator.
Sylgard Dissecting Plate: Mix gently (to avoid bubbles) 10:1in a beaker. Pour mixture into Petri dish (any size). Allow SylGard to cure overnight in 37C incubator.

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References

  1. Jan, L. Y., Jan, Y. N. Properties of the larval neuromuscular junction in Drosophila Melanogaster. J. Physiol. 262, 189-214 (1976).
  2. Johansen, J., Halpern, M. E., Johansen, K. M., Keshishian, H. Stereotypic morphology of glutamatergic synapses on identified muscle cells of Drosophila larvae. J Neurosci. 9, 710-725 (1989).
  3. Kesheshian, H., Broadie, K., Chiba, A., Bate, M. The Drosophila neuromuscular junction: a model system for studying synaptic development and function. Annu Rev Neurosci. 19, 545-575 (1996).
  4. Vactor, D. V., Sink, H., Fambrough, D., Tsoo, R., Goodman, C. S. Genes that control neuromuscular specificity in Drosophila. Cell. 73-1137 (1993).
  5. Feng, A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae. J Neurogenet. 18, (2), 377-402 (2004).

Comments

2 Comments

  1. Hi. I wonder if the fixation using formaldehyde is fine for any ulterior immunohistochemistry assay. Have you any experience if there is necessities for performing the fixation with other fixative? I´m asking this regarding the preservation of cytoskeletal proteins

    Reply
    Posted by: Anonymous
    February 13, 2009 - 4:07 PM
  2. Very well done Mr. Brent. You have a have steady hand !    

    Reply
    Posted by: Anonymous
    March 17, 2009 - 10:47 AM

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