Name: long-term time lapse imaging of mouse cochlear explants
Uploaded: 2014-11-02T15:00:00
Description: Watch this Scientific Journal Video about Long-term Time Lapse Imaging of Mouse Cochlear Explants at JoVE.com

谢谢孙威利对技术援助和克里斯Gellynck博士有益的意见和三个匿名评审的建设性意见。这项工作是由美国的新宁听力重建倡议

小鼠组织从Sox2的EGFP收获-reporter维护和安乐死按照中国的护理和使用实验动物的动物护理准则加拿大议会小鼠12。 1.培养胚胎耳蜗<ol><li>通过混合8.89毫升DMEM中，加入1ml的胎牛血清（FBS），100微升100×N2补充，和10μl的10mg / ml的环丙沙星补充的Dulbecco改良的Eagle培养基（DMEM）。通过混合495毫升的HBSS，用5毫升100％的HEPES补充Hank氏平衡盐溶液（HBSS）中。 </li><...

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+the+inner+ear+in+organ+culture.">Growth of the inner ear in organ culture.</a> The Annals of otology, rhinology, and laryngology. 83, 1-16 (1974).</li><li>Jacques, B. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dual+function+for+canonical+Wnt/beta-catenin+signaling+in+the+developing+mammalian+cochlea.">A dual function for canonical Wnt/beta-catenin signaling in the developing mammalian cochlea.</a> Development. 139, 4395-4404 (2012).</li><li>Castellano-Munoz, M., Peng, A. W., Salles, F. T., Ricci, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swept+field+laser+confocal+microscopy+for+enhanced+spatial+and+temporal+resolution+in+live-cell+imaging.+Microscopy+and+microanalysis+:+the+official+journal+of+Microscopy.">Swept field laser confocal microscopy for enhanced spatial and temporal resolution in live-cell imaging. Microscopy and microanalysis : the official journal of Microscopy.</a> Society of America, Microbeam Analysis Society, Microscopical Society of Canada. 18, 753-760 (2012).</li><li>Szarama, K. B., Gavara, N., Petralia, R. S., Chadwick, R. S., Kelley, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thyroid+hormone+increases+fibroblast+growth+factor+receptor+expression+and+disrupts+cell+mechanics+in+the+developing+organ+of+corti.">Thyroid hormone increases fibroblast growth factor receptor expression and disrupts cell mechanics in the developing organ of corti.</a> BMC developmental biology. 13, 6 (2013).</li><li>Appler, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gata3+is+a+critical+regulator+of+cochlear+wiring.">Gata3 is a critical regulator of cochlear wiring.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 33, 3679-3691 (2013).</li><li>Wibowo, I., Pinto-Teixeira, F., Satou, C., Higashijima, S., Lopez-Schier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalized+Notch+signaling+sustains+epithelial+mirror+symmetry.">Compartmentalized Notch signaling sustains epithelial mirror symmetry.</a> Development. 138, 1143-1152 (2011).</li><li>Hashimoto, S., Kato, N., Saeki, K., Morimoto, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+high-potential+embryos+by+culture+in+poly(dimethylsiloxane)+microwells+and+time-lapse+imaging.">Selection of high-potential embryos by culture in poly(dimethylsiloxane) microwells and time-lapse imaging.</a> Fertility and sterility. 97, 332-337 (2012).</li><li>Matsuoka, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology-based+prediction+of+osteogenic+differentiation+potential+of+human+mesenchymal+stem+cells.">Morphology-based prediction of osteogenic differentiation potential of human mesenchymal stem cells.</a> PloS one. 8, (2013).</li><li>Ma, G. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al.Transforming+growth+factor-beta1+and+-beta2+in+gastric+precancer+and+cancer+and+roles+in+tumor-cell+interactions+with+peripheral+blood+mononuclear+cells+in+vitro.">et al.Transforming growth factor-beta1 and -beta2 in gastric precancer and cancer and roles in tumor-cell interactions with peripheral blood mononuclear cells in vitro.</a> PloS one. 8, (2013).</li><li>Taranova, O. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOX2+is+a+dose-dependent+regulator+of+retinal+neural+progenitor+competence.">SOX2 is a dose-dependent regulator of retinal neural progenitor competence.</a> Genes &amp; development. 20, 1187-1202 (2006).</li><li>Chen, P., Segil, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p27(Kip1)+links+cell+proliferation+to+morphogenesis+in+the+developing+organ+of+Corti.">p27(Kip1) links cell proliferation to morphogenesis in the developing organ of Corti.</a> Development. , 1581-1590 (1999).</li></ol>

有几种技术要点当培养物被建立并在成立的时间推移显微镜为了长期成像来考虑。 我们用基底膜基质作为基质培养耳蜗植体，但在衬底应匹配于细胞类型。例如，将图像的神经元培养物，它可能是更好的是提供一种纤连蛋白涂层。温育温度和气体组成也应当根据组织类型选择。选择外植体的年龄是很重要的。我们开始对E13在最早因为对E12的生长方向是难以预料。因为它是必?...

哺乳动物耳蜗是高度有序的复杂器官。在小鼠中，从上一天E11耳泡和发育程序完成后，将原始内耳的出现在出生后早期阶段之间，细胞信号转导的多种波和协调的基因表达的变化发生。从基本运行到顶点的蜗管的是声音探测感觉上皮尔蒂的，或器官。柯蒂氏器的发展精美控制，使得通过显影结束时，其将包括内毛细胞的单排，三排外毛细胞的散布有五排支持细胞（两列支柱细胞，三行Deiters&#39;细胞）1。偏离这个精确的顺序会导致听力下降，heighlighting studye的国税发起源和感官epithemlium 2的图案化的重要性。 在胚胎小鼠COCH的体外培养LEA是研究Corti器的发展机制的一个重要工具。该技术成立于1974年，并在过去的40年中，一直被用来阐明许多由感官上皮规定和Corti器建立3的机制。耳蜗是一个复杂的器官的动态发育过程;一个操作可能需要长达7天的表现1。例如，添加GSK3β抑制剂在E13耳蜗植体培养时，最佳的潜伏期，观察该化合物的一个健壮效果为6天4。 显影耳蜗的实时成像可以调查变化的柯蒂氏器的形态，基因表达的变化，跟踪迁移的，增殖或死亡的细胞，并允许实时观察药剂的结果的和信号的中断途径。到现在为止，住耳蜗的摄像已经主要使用共聚焦显微镜对柯蒂氏在短时间内5-8的脏器图像的小区域进行的，但该技术具有由于外植体存活的局限性。在较长期的操作慢发育过程的影响成像，成像环境是至关重要的。典型的共焦实时成像系统采用的加湿塑料箱，它位于所述的显微镜。热和湿度，可以通过在孵育箱那里它满足显微镜台，通过接入窗，通过铰链孔，并通过围绕microscope-各部分的间隙的间隙逸出，如目标或光源。这不是最优的用于维持健康植超过两三天。 我们定义“孵化显微镜&#39;为倒置显微镜内部的标准的CO 2培养箱中密封，而不是在显微镜周围建造一个孵化器。孵化器显微镜前趋于实验的寿命，使得而非成像在两三天，样品可以被成像长达两周。培养箱显微镜提供用于细胞生长和分化良好的环境，以最小的干扰外植体培养物和标准控制的条件。在研究，发生在多天里，通常通过从培养箱中取出，并携带它们到一个倒置荧光显微镜诉诸成像样品，每天。虽然这种方法可以工作，从培养箱中取出盘子对其造成的敏感发展组织应力。由于除去从孵化器中的更改培养用培养基和波动的温度的酸度可导致次优的发展和不健康的组织。成象的区域在同一焦平面上，并在相同的取向，在每一个时间点是非常有挑战性的。通过利用恒温箱中的自动化系统，它能够维持健康组织，采集图像，在多个时间点，并确保在同一区域被捕获在每一帧。近年来几个集成显微镜组织培养箱被开发出来，这些不仅在临床实践9，而且在干细胞和癌症研究10,11是有用的。 在这里，我们提出了一个协议，用于胚胎小鼠耳蜗外植体的长期实时成像。我们使用自动显微镜系统内部的标准的CO 2培养箱具有捕获多个样品的图像在设定的时间点的能力。该系统由一个倒置显微镜孵化器内设定的。样品被放置在一个旋转的讲台，允许多个样品的成像在每个时间点。照明，图像采集和讲台的旋转是通过的Metamorph软件操作的自动化系统控制。通过操作软件设置的成像程序，我们可以设置一个实验，运行长达TWØ周以最少的人工干预。在这个例子中我们使用两个亮场和荧光显示大规模增长和耳蜗的重排，并且具体地，prosensory区域。在该实验中，耳蜗将从Sox2的EGFP报告小鼠解剖胚胎天E13。 在体外培养物将被建立，然后成像五天。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle media</td>
			<td>Gibco</td>
			<td>12430</td>
			<td>Multiple brands manufacture this</td>
			<td>http://www.lifetechnologies.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/classical-media/dmem.html</td>
		</tr>
		<tr>
			<td>Basement membrane extract&#160;</td>
			<td>Corning</td>
			<td>354230</td>
			<td>Matrigel. Alternative similar products are available from other suppliers. 
			http://catalog2.corning.com/Lifesciences/en-US/Shopping/Product.aspx?categoryname=Cell+Culture+and+Bioprocess%28Lifesciences%29|Extracellular+Matrix+Proteins+ECMs+and+Attachment+Factors%28Lifesciences%29|Matrigel+Basement+Membrane+Matrix+%28Lifesciences%29</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>16000044</td>
			<td>Multiple brands manufacture this 
			http://www.lifetechnologies.com/search/global/searchAction.action?query=fbs&#38;resultPage=1&#38;results PerPage=15&#38;autocomplete=</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>5630080</td>
			<td>Multiple brands manufacture this 
			http://www.lifetechnologies.com/search/global/searchAction.action?query=hepes&#38;resultPage=1&#38;results PerPage=15&#38;autocomplete=</td>
		</tr>
		<tr>
			<td>100 x N2 supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td>Multiple brands manufacture this 
			http://www.lifetechnologies.com/search/global/searchAction.action?query=n2&#38;resultPage=1&#38;results PerPage=15&#38;autocomplete=</td>
		</tr>
		<tr>
			<td>Ciprofloxacin</td>
			<td>Sigma Aldrich</td>
			<td>17850-5G-F</td>
			<td>Multiple brands manufacture this 
			http://www.sigmaaldrich.com/catalog/product/fluka/17850?lang=en&#38;region=CA</td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution</td>
			<td>Gibco</td>
			<td>14170161</td>
			<td>Multiple brands manufacture this. Should be refidgerated before use. 
			http://www.lifetechnologies.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/reagents/balanced-salt-solutions/hbss-hanks-balanced-salt-solution.html?s_kwcid=AL!3652!3!26107410508!e!!g!!hbss&#38;ef_id=xoFOglw2s UMAAMU8:20140228185720:s</td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Fine Science tools</td>
			<td>11254-20</td>
			<td>Size number 5 
			http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=350&#38;CategoryId=29</td>
		</tr>
		<tr>
			<td>Curette&#160;</td>
			<td>Fine Science Tools</td>
			<td>10080-05</td>
			<td>size 1 mm&#160; 
			http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=91&#38;CategoryId=118</td>
		</tr>
		<tr>
			<td>Insect Pins</td>
			<td>Fine Science Tools</td>
			<td>26001-35</td>
			<td>Must be stainless Steel 
			http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=124</td>
		</tr>
		<tr>
			<td>50 mm plastic dishes</td>
			<td>Corning/Falcon</td>
			<td>351006</td>
			<td>Multiple brands manufacture this</td>
		</tr>
		<tr>
			<td>charcoal</td>
			<td>Sigma Aldrich</td>
			<td>05105-250G</td>
			<td>Multiple brands manufacture similar items 
			http://www.sigmaaldrich.com/catalog/product/fluka/05105?lang=en&#38;region=CA</td>
		</tr>
		<tr>
			<td>184 silicone elastomer</td>
			<td>Dow/Corning&#160;</td>
			<td>SYLGARD&#174; 184 SILICONE ELASTOMER KIT</td>
			<td>Dishes are home made several weeks in advance.&#160; Silicone elastomer can be from any supplier.<a href="http://www.dowcorning.com/applications/search/products/Details.aspx?prod=01064291">http://www.dowcorning.com/applications/search/products/Details.aspx?prod=01064291</a></td>
		</tr>
		<tr>
			<td>Glass bottom dishes</td>
			<td>MatTek</td>
			<td>P35G-0-10-C</td>
			<td>The dimensions of the dish are determined by the specifications of the imaging system.&#160; 35 mm diameter, 10mm well, number 0 coverslip fits Olympus Vivaview FL. 
			http://glass-bottom-dishes.com/catalog/index.php?main_page=product_info&#38;cPath= 1_4_15&#38;products_id=2</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Zeiss&#160;</td>
			<td>495101-9804-000</td>
			<td>&#160;Stemi 2000 model. multiple brands manufacture similar items 
			http://microscopy.zeiss.com/microscopy/en_de/products/stereo-zoom-microscopes/stemi-2000.html</td>
		</tr>
		<tr>
			<td>Cold Light Source</td>
			<td>Zeiss</td>
			<td>000000-1063-182</td>
			<td>Multiple brands manufacture similar items 
			http://microscopy.zeiss.com/microscopy/en_de/products/microscope-components/lightsources.html</td>
		</tr>
		<tr>
			<td>Fluorescent Stereomicroscope&#160;</td>
			<td>Leica microsystems</td>
			<td>Contact Leica microsystems</td>
			<td>Leica M165-FC. Multiple brands manufacture similar items 
			http://www.leica-microsystems.com/products/stereo-microscopes-macroscopes/fluorescence/details/product/leica-m165-fc/</td>
		</tr>
		<tr>
			<td>Incubator Microscope +imaging software</td>
			<td>Olympus</td>
			<td>Contact Olympus</td>
			<td>Inverted microcope sealed inside a Co2 incubator. Vivaview FL incubator microscope with proprietry Metamorpoh imaging software. 
			http://olympuscanada.com/seg_section/product.asp?product=1055&#38;c=0</td>
		</tr>
		<tr>
			<td>Clean bench</td>
			<td>Thermo Scientific</td>
			<td>51029701</td>
			<td>Multiple brands manufacture similar items 
			http://www.thermoscientific.com/en/product/heraguard-eco-clean-bench.html</td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo Scientific</td>
			<td>3310</td>
			<td>Multiple brands manufacture similar items 
			http://www.thermoscientific.com/en/products/co2-incubators.html</td>
		</tr>
		<tr>
			<td>Laminar Flow hood</td>
			<td>Thermo Scientific</td>
			<td>51026651</td>
			<td>Multiple brands manufacture similar items 
			http://www.thermoscientific.com/en/products/biological-safety-cabinets-clean-benches.html</td>
		</tr>
	</tbody>
</table>

long-term time lapse imaging of mouse cochlear explants

在这里，我们提出了活鼠胚胎的耳蜗植长期时间推移成像的方法。负责建设的哺乳动物耳蜗收益的高度有序的，复杂的结构，大约十天后的发展计划。为了研究在此期间基因表达的变化和它们的响应的药物或基因操作，长期成像是必要的。先前，实时成像已经典型地通过移植组织的存活，在湿润的腔室顶上标准显微镜的限制。难以保持对培养物生长有关的湿度和温度的最佳条件已经放置在成像实验的长度的限制。显微镜集成到修改后的组织培养孵化器提供长期，实时成像的良好环境。在这个方法中，我们演示了如何建立胚胎小鼠耳蜗植，以及如何使用孵化器的显微镜来进行时间的推移意马使用毫微都亮场和荧光显微镜检查的典型胚胎天（E） - 13耳蜗植体和Sox2，耳蜗的prosensory细胞的标记，在5天的行为。

在这里，我们将展示一个蒙太奇（ 图1），看电影（ 图2），这说明一个典型的器官耳蜗植将如何发展，如果镀上E13.5。一个Sox2的记者鼠标采用可视化的prosensory区域。动画示出了耳蜗经历生长和收敛性和扩展，使细胞中的绿色Sox2的域的横向区域似乎没有划分为围绕它展开的组织。这是Corti器的特性;关于E13准感觉上皮退出细胞周期和随后称为防扩散13?...

Watch this Scientific Journal Video about Long-term Time Lapse Imaging of Mouse Cochlear Explants at JoVE.com

Long-term Time Lapse Imaging of Mouse Cochlear Explants

胚胎哺乳动物耳蜗的实时成像是具有挑战性的，因为手头的发育过程超过十天时间梯度进行操作。在这里，我们提出了培养，然后从成像荧光记者鼠标采取了五天的胚胎耳蜗植体组织的方法。

老鼠耳蜗外植体的长期时间推移成像

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Research

JoVE Journal

Neuroscience

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 ...

科研

神经科学

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

在发展中国家斑马鱼的前脑的神经祖细胞克隆相关的定时实时成像

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

抛光和钢筋，颅骨变薄窗口，为长期的小鼠脑成像

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

急性片的成年小鼠前脑的神经细胞迁移的延时成像

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

成神经细胞迁移的小鼠急性脑片在体内电穿孔产后和时间推移成像

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

琼脂糖微室的长期钙成像<em&gt;秀丽隐杆线虫</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

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 ...

Generation and Long-term Maintenance of Nerve-free Hydra

Generation and Long-term Maintenance of Nerve-free Hydra

无神经的一代和长期维持<em&gt;水润</em

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

成人脑切片长期抑郁症诱导评估

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

长期，无血清培养有机小鼠视网膜外植与完整的视网膜色素上皮

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of ...