Name: organotypic slice culture of gfp-expressing mouse embryos for real-time imaging of peripheral nerve outgrowth
Uploaded: 2011-03-29T11:00:00
Description: Watch this Scientific Journal Video about Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth at JoVE.com

作者承认的想法执行后，小鼠胚胎5片文化的原始来源。我们要感谢我们gofer在拍摄约阿希姆基尔希的慷慨科学支持和安娜德劲。这项工作是由德国研究基金会（德意志研究联合会：Sonderforschungsbereich 488 Teilprojekt B7/B9）和海德堡大学（卓越集群无线通信网络）。

第1部分：准备切片和培养。 <ol><li>切片培养基（DMEM培养液，25％1X HBSS中，25％胎牛血清，0.5％葡萄糖，1毫米谷氨酰胺​​，2.5毫米的HEPES，pH值7.3）和3厘米Millicell小CM 0.4微米的文化准备10厘米的组织培养板膜插入和孵化器保持在37 ° C和5％的CO 2。 </li><li>在微波炉中加热4％，熔点低，在PBS点琼脂糖，并继续加热板，使其保持在约37 ° C。 </li><li>填补10厘米的细菌培养皿中，用PBS（140毫米氯...

<ol>
	<li>Tucker, K. L., Meyer, M., Barde, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophins+are+required+for+nerve+growth+during+development.">Neurotrophins are required for nerve growth during development.</a> Nat Neurosci. 4, 29-37 (2001).</li><li>Brachmann, I., Jakubick, V. C., Shaked, M., Unsicker, K., Tucker, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+slice+culture+system+for+the+imaging+of+nerve+development+in+embryonic+mouse.">A simple slice culture system for the imaging of nerve development in embryonic mouse.</a> Dev Dyn. 236, 3514-3523 (2007).</li><li>Collingridge, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+brain+slice+preparation:+a+tribute+to+the+pioneer+Henry+McIlwain.">The brain slice preparation: a tribute to the pioneer Henry McIlwain.</a> Journal of neuroscience methods. 59, 5-9 (1995).</li><li>Katz, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+circuitry+of+identified+projection+neurons+in+cat+visual+cortex+brain+slices.">Local circuitry of identified projection neurons in cat visual cortex brain slices.</a> J Neurosci. 7, 1223-1249 (1987).</li><li>Hotary, K. B., Landmesser, L. T., Tosney, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo+slices.">Embryo slices.</a> Methods Cell Biol. 51, 109-124 (1996).</li></ol>

在比较广泛的方法，准备妊娠中期的小鼠胚胎的胚胎切片文化（E10 - E12），我们观察到一个vibratome毫无疑问，尊重文化的整体可行性和重复性生产的最可靠的结果神经生长模式。相比之下，使用斩波器3被证明是完全inviable一个McIlwain组织切片准备。我们原任一个断头台的方法4，在整个胚胎的垃圾可以同时缠一堑长一连续篦生产400 微米的第1钨丝切片准备。虽然这种技术已?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>HBSS 10x</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>14180</td>
<td>&nbsp;</td>
<tr>
<td>Dissection tools</td>
<td>&nbsp;</td>
<td>FST</td>
<td>various</td>
<td>&nbsp;</td>
<tr>
<td>L.M.P. agarose</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15517-022</td>
<td>&nbsp;</td>
<tr>
<td>Whatmann paper</td>
<td>&nbsp;</td>
<td>Whatman Int.</td>
<td>3030917</td>
<td>&nbsp;</td>
<tr>
<td>Shortened firepolished pipettes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>41966</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>10270-106</td>
<td>&nbsp;</td>
<tr>
<td>Pen Strep</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine 100x</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Vibratome</td>
<td>&nbsp;</td>
<td>Microm International GmbH</td>
<td>HM 650 V</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>BX61WI</td>
<td>&nbsp;</td>
<tr>
<td>analySIS</td>
<td>&nbsp;</td>
<td>Soft Imaging System</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Millicell-CM inserts</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>PICMORG 50</td>
<td>&nbsp;</td>
<tr>
<td>10 cm culture plates</td>
<td>&nbsp;</td>
<td>Greiner Bio-One</td>
<td>633171</td>
<td>&nbsp;</td>
<tr>
<td>LOCTITE 406</td>
<td>&nbsp;</td>
<td>Henkel</td>
<td>142580</td>
<td>&nbsp;</td>
<tr>
<td>Razor blades</td>
<td>&nbsp;</td>
<td>Thermo Fisher</td>
<td>none</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>SMZ800</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>9105.2</td>
<td>&nbsp;</td>
<tr>
<td>Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G7021</td>
<td>&nbsp;</td>
<tr>
<td>x4 objective</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>PL series</td>
<td>&nbsp;</td>
<tr>
<td>x10 objective</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>UPLFL &#x2013;PH series</td>
<td>&nbsp;</td>
<tr>
<td>Filter</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>U-MNIBA2</td>
<td>&nbsp;</td>
<tr>
<td>CCD camera</td>
<td>&nbsp;</td>
<td>Soft Imaging System</td>
<td>SIS F-View II</td>
<td>&nbsp;</td>
<tr>
<td>Equipment for heated chamber</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>CTI-Controller 3700 and incubator S #11531171</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

organotypic slice culture of gfp-expressing mouse embryos for real-time imaging of peripheral nerve outgrowth

多种用途，小鼠胚胎体外培养器官切片是可取的。例如，我们采用了转基因小鼠线（tauGFP）在绿色荧光蛋白（EGFP）的增强版本是专门表示，在发展的中枢和外周神经系统1所有神经元的可能性，让这两个电影的支配前肢和操纵这个过程中，药理学和基因工程技术生产的 2 。切片文化的培育成功，最关键的参数，是这片准备的方法。多种方法进行广泛的测试后，我们发现一个vibratome设备切片胚胎等，他们经常在文化，展示了可行性的几天内，最重要的结果是最好的，在这样一个时代的发展特定的方式。对于妊娠中期的胚胎，这包括脊神经从脊髓和背根神经节，他们在外围和适当的骨骼和肌肉组织的决心目标的正常生长。 在这项工作中，我们提出了一种用于加工成300整个胚胎的胚胎一天（五）E10的E12的方法 - 种植400微米片在一个标准的组织文化的孵化器，可研究后切片准备两天。这种方法成功的关键是使用vibratome每个琼脂糖包埋胚胎切片。其次是种植后Millicell小文化膜插入后放在一个中等体积小，接口中的文化技术的切片。凋落物平均7胚胎常规生产，至少有14片（2-3片每胚胎前肢地区），由于胚胎的年龄以及切片厚度略有不同。大约80％的培养切片显示神经生长，它可以测量througout培养期2。使用tauGFP鼠标线的代表结果表明。

Watch this Scientific Journal Video about Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth at JoVE.com

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

我们现在准备妊娠中期的小鼠胚胎的器官切片周围神经生长的培养和时间推移成像的方法。

器官型切片文化绿色荧光蛋白表达的实时成像周围神经生长的小鼠胚胎

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

Research

JoVE Journal

Neuroscience

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

海马脑片的器官型检查神经元损伤的文化模式

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

科研

神经科学

Organotypic Hippocampal Slice Cultures

器官型海马切片培养

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

从大脑皮层的器官型切片制备全细胞记录

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

在完整的视网膜电生理特性表达GFP的细胞群

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

实时 fMRI 神经训练管理协议

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

延期共聚焦显像移植神经元在器官性切片培养胚胎小鼠脑使用<em&gt;在Utero</em&gt;电穿孔

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

前子宫内胚胎小鼠脑电穿孔和脊髓切片培养迁移 GABAergic 中间神经元的活体成像

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

髓鞘有机小脑片培养的制备及免疫染色

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...