欢迎您, 登录 | 注册

首页 | English

X
加载中

This technique will allow using brain slices to study several aspects of cortical development (i.e. neurogenesis), as well as neuronal differentiation (i.e. neuronal migration, axon and dendrite formation) in situ. This protocol is suitable for various embryonic stages (Calderon de Anda et al., 2010; Ge et al., 2010).

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

Organotypic Slice Culture of Embryonic Brain Sections
胚胎脑切片的培养

发育生物学 > 细胞生长和命运决定 > 神经元
作者: Froylan Calderon de Anda
Froylan Calderon de AndaAffiliation: Center For Molecular Neurobiology Hamburg (ZMNH), University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany
For correspondence: froylan.calderon@zmnh.uni-hamburg.de
Bio-protocol author page: a227
Vol 3, Iss 3, 2/5/2013, 7728 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.327

[Abstract] This technique will allow using brain slices to study several aspects of cortical development (i.e. neurogenesis), as well as neuronal differentiation (i.e. neuronal migration, axon and dendrite formation) in situ. This protocol is suitable for various embryonic stages (Calderon de Anda et al., 2010; Ge et al., 2010).
Keywords: Neuronal development(神经元发育), Cortical development(皮质发育), Neuronal migration(神经元的迁移)

[Abstract] 该实验方案的中文版正在准备中...

Materials and Reagents

  1. Agarose Type VII (low melting) (Sigma-Aldrich, catalog number: A9045-100G )
  2. Neurobasal (Life Technologies, Gibco®, catalog number: 21103-049 )
  3. B27 (Life Technologies, InvitrogenTM, catalog number: 17504-04 )
  4. Glutamine (Life Technologies, Gibco®, catalog number: 25030 )
  5. Penicillin/streptomycin (Sigma-Aldrich, catalog number: P4458 )
  6. Horse serum (Life Technologies, InvitrogenTM, catalog number: 16050-122 )
  7. N2 (Life Technologies, InvitrogenTM, catalog number: 17502-048 )
  8. 1x PBS
  9. Krazyglue or similar glue

Equipment

  1. Vibratome (Leica, model: VT1000 S)
  2. Millicell, sterilized culture plate insert (EMD Millipore)
  3. Dissecting tools (Ring forceps, Serrated forceps, Fine scissors)
  4. Curved spatula
  5. 30 mm petri dish

Procedure

  1. Remove the head of electroporated mouse embryos (see “In utero Electroporation of Mouse Embryo Brains” (Ge, 2011) and place it into ice-cold 1x PBS.
  2. Dissect out the whole brain and place it into ice-cold PBS. Do not let the brain more that 30 min in ice-cold PBS. Faster the brain is processed; better brain slices quality will be obtained.
  3. Fill a 30 mm petri dish with 3% low-melting agarose prepared in 1x PBS (use enough agarose to cover the brain) pre-warmed at 40 °C in a microwave. Monitor the temperature with a thermometer while steering the agarose solution with it. The brain will be put into the agarose when the temperature drops to about 35 °C-37 °C, try to use fresh 3% low-melting agarose every time you prepare brain slices.
  4. Take out the brain from the ice-cold PBS with a curved spatula and carefully remove the PBS solution from the tissue with a Kimwipe. Use the Kimwipe as a wick. Critical step to maintain the structure of the brain in culture: Try to remove as much PBS solution as possible from the tissue. Avoid the Kimwipe to adhere to the brain in order to prevent tissue damage. Place the Kimwipe as close as you can to the brain to absorb, as much PBS solution as possible. A “PBS solution-free” brain is better embedded into the agarose solution. Thus, once the brain is sliced the brain sections do not detach from the agarose.
  5. Place the brain into 3% low-melting agarose (35 °C-37 °C) in a petri dish (olfactory bulb facing down, Figure 1A).
  6. Put the petri dish on ice and wait until the agarose solidifies. Carefully keep the position of the brain with the spatula meanwhile the agarose solidifies.
  7. Cut the agarose around the brain and keep the brain in the middle of the agarose block. Try to cut the block with a pyramidal shape in such a way that the base of the brain (cerebellum) is the base of the pyramid and the olfactory bulb faces the apex of the pyramid (Figure 1B). This agarose block configuration will allow a steady position of the block when glued at the vibratome tray’s holder (the base of the agarose pyramid faces the vibratome holder).
  8. Glue the agarose block containing the brain onto the vibratome tray with Krazyglue or similar glue.
  9. Add ice-cold 1x PBS into the vibratome tray to cover the agarose block.
  10. Slices the agarose block. Slice thickness: 240 μm (tissue flatten over time with thinner slices). Vibratome settings: speed 4-5; vibration 6.
  11. Collect slices with curved spatula carefully to avoid the tissue to detach from the agarose. Transfer the slices into a Millicell culture insert system containing 1 ml medium (Figure C; Neurobasal supplemented with B27, N2, 0.5 mM Glutamine, 1% penicillin/ streptomycin, and 5% horse serum). Put as many slices as you can put on top of the membrane insert (generally 4-5 slices per insert).
  12. Remove carefully the solution that surrounds the slice with pipette tip. Tissue must be exposed to air.
  13. Place the culture insert dish with slices into tissue culture incubator (37 °C) for 1-2 h
    The slices are ready to use (i.e. Time-lapse imaging). This culture system has been used for 48 h time-lapse imaging (de Anda F., et al., 2010; Ge et al., 2010). Also is possible to use this culture system for pharmacological treatments (de Anda F., et al., 2010; Ge et al., 2010).



    Figure 1. (A) Brain is embedded, with the olfactory bulb facing down, in a preti dish with 3% low-melting agarose. (B) Once the agarose solidifies cut the agarose around the brain with a pyramidal shape in such a way that the base of the brains (side of the cerebellum) is at the base of the pyramid and olfactory bulb faces the apex of the pyramid. (C) Transfer coronal sections from the brain on top of the Millicell culture insert system containing 1 ml medium.

Recipes

  1. Neurobasal (250 ml)
    50x B27
    4.5 ml
    200 mM Glutamine
    2.5 ml
    50x Penicillin/streptomycin
    2.5 ml
    Horse serum
    12.5 ml
    100x N2
    2.5 ml (it should be added to the medium the day of the experiment; 10 μl/1 ml medium)
    Total
    24.5 ml/250 ml Neurobasal
    This media can be stored at 4 °C protected from light for several months

Acknowledgments

This protocol was adapted form the previously published paper: Calderon de Anda et al. (2010). C.d.A.F. was supported by a postdoctoral fellowship from the Simons Initiative on Autism and the Brain Infrastructure Grant Program.

References

  1. Calderon de Anda F., Meletis, K., Ge, X., Rei, D. and Tsai, L. H. (2010). Centrosome motility is essential for initial axon formation in the neocortex. J Neurosci 30(31): 10391-10406.
  2. Calderon de Anda F., Rosario, A. L., Durak, O., Tran, T., Graff, J., Meletis, K., Rei, D., Soda, T., Madabhushi, R., Ginty, D. D., Kolodkin, A. L. and Tsai, L. H. (2012). Autism spectrum disorder susceptibility gene TAOK2 affects basal dendrite formation in the neocortex. Nat Neurosci 15(7): 1022-1031.
  3. Ge, X. (2012). In utero Eletroporation of Mouse Embryo Brains. Bio-protocol 2(13): e231. 
  4. Ge, X., Frank, C. L., Calderon de Anda, F. and Tsai, L. H. (2010). Hook3 interacts with PCM1 to regulate pericentriolar material assembly and the timing of neurogenesis. Neuron 65(2): 191-203.

材料和试剂

  1. 琼脂糖VII型(低熔点)(Sigma-Aldrich,目录号:A9045-100G)
  2. Neurobasal(Life Technologies,  Gibco ®,目录号:21103-049)
  3. B27(Life Technologies,Invitrogen TM ,目录号:17504-04)
  4. 谷氨酰胺(Life Technologies,< Gibco ,目录号:25030)
  5. 青霉素/链霉素(Sigma-Aldrich,目录号:P4458)
  6. 马血清(Life Technologies,Invitrogen TM,目录号:16050-122)
  7. N2(Life Technologies,Invitrogen TM,目录号:17502-048)
  8. 1x PBS
  9. Krazyglue或类似的胶水

设备

  1. Vibratome(Leica,型号:VT1000 S)
  2. Millicell,灭菌培养板插入物(EMD Millipore)
  3. 解剖工具(环形钳,锯齿镊子,细剪)
  4. 弯曲的小铲
  5. 30 mm培养皿

程序

  1. 移除电穿孔小鼠胚胎的头部(请参阅" 在子宫内电穿孔小鼠胚胎脑57"(Ge,2011),并将其置于冰冷的1x PBS中。
  2. 解剖整个大脑,并将其放入冰冷的PBS。不要让大脑在冰冷的PBS中30分钟。更快地处理大脑;将获得更好的脑切片质量
  3. 在30℃培养皿中加入在1×PBS中制备的3%低熔点琼脂糖(使用足够的琼脂糖覆盖脑),在微波中在40℃预热。用温度计监测温度,同时用它控制琼脂糖溶液。当温度降至约35°C-37°C时,大脑将被放入琼脂糖中,每次准备脑切片时,尝试使用新鲜的3%低熔点琼脂糖。
  4. 用弯曲的刮刀从冰冷的PBS中取出大脑,用Kimwipe小心地从组织中取出PBS溶液。使用Kimwipe作为灯芯。保持培养中大脑结构的关键步骤:尝试从组织中尽可能多地除去PBS溶液。避免Kimwipe粘附到大脑,以防止组织损伤。放置Kimwipe尽可能接近,你可以到大脑吸收尽可能多的PBS溶液。 "无PBS溶液"的脑更好地嵌入琼脂糖溶液中。因此,一旦脑切片,脑切片不从琼脂糖上分离。
  5. 将大脑放入3%低熔点琼脂糖(35°C-37°C)在陪替氏培养皿(嗅球面朝下,图1A)。
  6. 把培养皿放在冰上,等待琼脂糖凝固。小心地用刮刀保持大脑的位置,同时琼脂糖凝固。
  7. 剪切大脑周围的琼脂糖,保持大脑在琼脂糖块的中间。尝试切割金字塔形状的块,使得大脑(小脑)的基部是金字塔的底部,并且嗅球面对金字塔的顶点(图1B)。这种琼脂糖块配置将允许块在胶合在vibratome托盘的支架(琼脂糖金字塔的基部面向vibratome支架)的稳定位置。
  8. 用Krazyglue或类似的胶将含有大脑的琼脂糖块胶接到vibratome托盘上
  9. 将冰冷的1x PBS加入vibratome托盘以覆盖琼脂糖块。
  10. 切琼脂糖块。切片厚度:240μm(随着时间的推移,薄片更薄的组织)。振动器设置:速度4-5;振动6.
  11. 小心收集片用弯曲的小铲,以避免组织从琼脂糖分离。将切片转移到含有1ml培养基(图C;补充有B27,N2,0.5mM谷氨酰胺,1%青霉素/链霉素和5%马血清的Neurobasal)的Millicell培养插入系统中。放入尽可能多的切片,你可以放在膜插入物的顶部(通常4-5片每个插入)。
  12. 用移液管吸头小心地移除切片周围的溶液。组织必须暴露于空气中。
  13. 将培养插入皿与切片放入组织培养孵化器(37℃)1-2小时
    切片准备使用(即延时成像)。该培养系统已用于48小时时间推移成像(de Anda F.,et al。,2010; Ge et al。,2010)。也可以使用这种培养系统用于药理学治疗(de Anda F.,et al。,2010; Ge等人,2010)。



    图1. (A)大脑是嵌入的,嗅球面朝下,在3%低熔点琼脂糖的preti菜。 (B)一旦琼脂糖凝固,将大脑周围的琼脂糖切成锥体形状,使得大脑底部(小脑的侧面)位于金字塔的底部,嗅球面向金字塔的顶点。 (C)从包含1ml培养基的Millicell培养插入系统顶部的脑转移冠状切片。

食谱

  1. Neurobasal(250ml)
    50x B27
    4.5 ml
    200mM谷氨酰胺 2.5 ml
    50x青霉素/链霉素 2.5 ml
    马血清
    12.5 ml
    100x N <2>
    2.5ml(应该在实验当天加入培养基;10μl/1ml培养基)
    总计
    24.5ml/250ml Neurobasal
    该介质可以在4℃避光保存几个月。

致谢

该协议改编自以前发表的论文:Calderon de Anda等人(2010)。 C.d.A.F.得到了Simons自闭症倡议和大脑基础设施赠款计划的博士后研究支持。

参考文献

  1. Calderon de Anda F.,Meletis,K.,Ge,X.,Rei,D.and Tsai,L.H。(2010)。 中枢运动对于新大脑皮质中初始轴突形成至关重要。 30(31):10391-10406。
  2. Calderon de Anda F.,Rosario,AL,Durak,O.,Tran,T.,Graff,J.,Meletis,K.,Rei,D.,Soda,T.,Madabhushi,R.,Ginty,DD,Kolodkin ,AL和Tsai,LH(2012)。 自闭症谱系障碍易感基因TAOK2 会影响新大脑皮质中的基底树突形成。 Nat Neurosci 15(7):1022-1031。
  3. Ge,X。(2012)。 在子宫内小鼠胚胎脑的电穿孔 生物协议 2(13):e231。 
  4. Ge,X.,Frank,C.L.,Calderon de Anda,F.and Tsai,L.H。(2010)。 Hook3与PCM1相互作用以调节近中心材料装配和神经发生的时机。 Neuron 65(2):191-203。
English
中文翻译

免责声明

为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。

X


How to cite this protocol: Anda, F. C. (2013). Organotypic Slice Culture of Embryonic Brain Sections. Bio-protocol 3(3): e327. DOI: 10.21769/BioProtoc.327; Full Text



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册
引用格式
分享
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook