Name: purkinje cell survival in organotypic cerebellar slice cultures
Uploaded: 2019-12-18T12:00:00
Description: Watch this Scientific Journal Video about Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures at JoVE.com

成像工作在西北大学高级显微镜中心进行，由NCI CCSG P30 CA060553慷慨支持，授予罗伯特·赫·卢里综合癌症中心。我们感谢肖恩·麦克德莫特的技术援助和支持，感谢玛雅·爱泼斯坦的手工绘制的插图，如图1所示。

所有涉及动物的实验都是根据西北大学动物研究委员会进行的。
1. 在器官小脑切片培养之前的准备
<ol><li>在事先喷洒 70% 乙醇的细胞培养罩中，在连接到无菌瓶接收器的 250 mL 瓶顶真空过滤器中制备 200 mL 培养基。加入100 mL的底中鹰（BME），50 mL的汉克斯平衡盐溶液（HBSS），50 mL的热灭活马血清，1 mL的200 mM L-谷氨酰胺（最终浓度1 mM），和5 mL的200克/升葡萄糖（最终浓度5毫克/?...

<ol>
	<li>Humpel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+brain+slice+cultures:+A+review.">Organotypic brain slice cultures: A review.</a> Neuroscience. 305, 86-98 (2015).</li><li>Gahwiler, B. 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S., Sotelo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purkinje+cell+survival+and+axonal+regeneration+are+age+dependent:+an+in+vitro+study.">Purkinje cell survival and axonal regeneration are age dependent: an in vitro study.</a> Journal of Neuroscience. 17 (10), 3710-3726 (1997).</li><li>Wiegreffe, C., Feldmann, S., Gaessler, S., Britsch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.</a> Journal of Visualized Experiments. (125), e55886 (2017).</li><li>Daviaud, N., 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=Modeling+nigrostriatal+degeneration+in+organotypic+cultures,+a+new+ex+vivo+model+of+Parkinson's+disease.">Modeling nigrostriatal degeneration in organotypic cultures, a new ex vivo model of Parkinson's disease.</a> Neuroscience. 256, 10-22 (2014).</li><li>Ghoumari, A. 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=Mifepristone+(RU486)+protects+Purkinje+cells+from+cell+death+in+organotypic+slice+cultures+of+postnatal+rat+and+mouse+cerebellum.">Mifepristone (RU486) protects Purkinje cells from cell death in organotypic slice cultures of postnatal rat and mouse cerebellum.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 (13), 7953-7958 (2003).</li><li>Labombarda, 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=Neuroprotection+by+steroids+after+neurotrauma+in+organotypic+spinal+cord+cultures:+a+key+role+for+progesterone+receptors+and+steroidal+modulators+of+GABA(A)+receptors.">Neuroprotection by steroids after neurotrauma in organotypic spinal cord cultures: a key role for progesterone receptors and steroidal modulators of GABA(A) receptors.</a> Neuropharmacology. 71, 46-55 (2013).</li><li>Gao, P., 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=P2X7+receptor-sensitivity+of+astrocytes+and+neurons+in+the+substantia+gelatinosa+of+organotypic+spinal+cord+slices+of+the+mouse+depends+on+the+length+of+the+culture+period.">P2X7 receptor-sensitivity of astrocytes and neurons in the substantia gelatinosa of organotypic spinal cord slices of the mouse depends on the length of the culture period.</a> Neuroscience. , 195-207 (2017).</li><li>Sundstrom, L., Morrison, B., Bradley, M., Pringle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+cultures+as+tools+for+functional+screening+in+the+CNS.">Organotypic cultures as tools for functional screening in the CNS.</a> Drug Discovery Today. 10 (14), 993-1000 (2005).</li><li>Jankowski, J., Miething, A., Schilling, K., Baader, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+purkinje+cell+death+is+spatiotemporally+organized+in+the+developing+mouse+cerebellum.">Physiological purkinje cell death is spatiotemporally organized in the developing mouse cerebellum.</a> Cerebellum. 8 (3), 277-290 (2009).</li><li>Ghoumari, A. M., Wehrle, R., Bernard, O., Sotelo, C., Dusart, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implication+of+Bcl-2+and+Caspase-3+in+age-related+Purkinje+cell+death+in+murine+organotypic+culture:+an+in+vitro+model+to+study+apoptosis.">Implication of Bcl-2 and Caspase-3 in age-related Purkinje cell death in murine organotypic culture: an in vitro model to study apoptosis.</a> European Journal of Neuroscience. 12 (8), 2935-2949 (2000).</li><li>Ghoumari, A. M., Wehrle, R., De Zeeuw, C. I., Sotelo, C., Dusart, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+protein+kinase+C+prevents+Purkinje+cell+death+but+does+not+affect+axonal+regeneration.">Inhibition of protein kinase C prevents Purkinje cell death but does not affect axonal regeneration.</a> Journal of Neuroscience. 22 (9), 3531-3542 (2002).</li><li>Ghoumari, A. 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=Neuroprotective+effect+of+mifepristone+involves+neuron+depolarization.">Neuroprotective effect of mifepristone involves neuron depolarization.</a> FASEB Journal. 20 (9), 1377-1386 (2006).</li><li>Repici, M., Wehrle, R., Antoniou, X., Borsello, T., Dusart, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=c-Jun+N-terminal+kinase+(JNK)+and+p38+play+different+roles+in+age-related+Purkinje+cell+death+in+murine+organotypic+culture.">c-Jun N-terminal kinase (JNK) and p38 play different roles in age-related Purkinje cell death in murine organotypic culture.</a> Cerebellum. 10 (2), 281-290 (2011).</li><li>Rakotomamonjy, J., Ghoumari, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-Derived+Neurotrophic+Factor+Is+Required+for+the+Neuroprotective+Effect+of+Mifepristone+on+Immature+Purkinje+Cells+in+Cerebellar+Slice+Culture.">Brain-Derived Neurotrophic Factor Is Required for the Neuroprotective Effect of Mifepristone on Immature Purkinje Cells in Cerebellar Slice Culture.</a> International Journal of Molecular Sciences. 20 (2), (2019).</li><li>Stoppini, L., Buchs, P. A., Muller, D. <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+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> Journal of Neuroscience Methods. 37 (2), 173-182 (1991).</li><li>Linkert, 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=Metadata+matters:+access+to+image+data+in+the+real+world.">Metadata matters: access to image data in the real world.</a> Journal of Cell Biology. 189 (5), 777-782 (2010).</li><li>Uesaka, N., 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=Organotypic+coculture+preparation+for+the+study+of+developmental+synapse+elimination+in+mammalian+brain.">Organotypic coculture preparation for the study of developmental synapse elimination in mammalian brain.</a> Journal of Neuroscience. 32 (34), 11657-11670 (2012).</li><li>Falsig, J., 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=Prion+pathogenesis+is+faithfully+reproduced+in+cerebellar+organotypic+slice+cultures.">Prion pathogenesis is faithfully reproduced in cerebellar organotypic slice cultures.</a> PLoS Pathogens. 8 (11), 1002985 (2012).</li><li>Bouslama-Oueghlani, L., Wehrle, R., Sotelo, C., Dusart, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+loss+of+the+ability+of+Purkinje+cells+to+regenerate+their+axons+occurs+in+the+absence+of+myelin:+an+in+vitro+model+to+prevent+myelination.">The developmental loss of the ability of Purkinje cells to regenerate their axons occurs in the absence of myelin: an in vitro model to prevent myelination.</a> Journal of Neuroscience. 23 (23), 8318-8329 (2003).</li><li>Apuschkin, M., Ougaard, M., Rekling, J. 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切贝拉尔切片培养是研究产后发育过程中程序化的Purkinje细胞死亡的有力工具。该技术可用于快速筛选候选分子的神经保护电位。主要优点是设置简单且经济高效，只需对设备进行适度投资（振动器的成本是纸巾切碎器的 3 倍）。此外，从一只小鼠小狗可以生成10到15个健康的切片，允许使用一种动物并行进行不同的检测。
为了获得一致且可重复的结果，尽可能高效地执行文?...

体外建模是生物医学研究的重要工具。它允许调查人员研究和严格控制受限制细胞类型或隔离系统/器官中的特定机制。组织切片培养是一种广泛应用的体外技术，特别是在神经科学领域。该方法最初由Göhwiler建立，他使用辊管技术2培养脑切片，后来由山本等人修改，他介绍了使用微孔膜进行皮质切片培养3。与原发细胞培养相比，有机切片培养具有保存组织细胞结构以及组织部分平面中的原生细胞-细胞连接的优势。
有机切片是从中枢神经系统的许多部位培养的，如海马体4，皮层5，纹状体6，小脑4，7，脊髓8，9等。它们已被证明是药物发现研究中的有力工具。神经活性分子的影响可以通过多种方式进行评估：使用免疫染色和生物化学分析的生存和神经退化，神经元回路形成，或使用电生理学和活成像中断。
这项工作的目的是描述一种简单的方法来执行器官小脑切片培养，这是已知的一个相关的模型，以模拟在体外小脑发育。特别是，我们专注于Purkinje细胞发育死亡的研究。在体内，Purkinje细胞在第一个产后周发生凋亡，在产后第3天（P3）11达到峰值。在小脑切片培养中也观察到同样的模式，当从P1和P8之间的动物身上取走脑细胞后，Purkinje神经元死于凋亡，峰值为P34，12。使用器官小脑切片培养物已经允许识别几个神经保护分子7，13，以及了解参与这个程序细胞死亡14，15，16的一部分机制。在这里，我们描述了一个基于在海马区Stoppini等人17号的研究，并适应小脑的Dusart等人的协议4它包括快速解剖和切除了产后脑切除术;将培养物切片到含有微孔膜的细胞培养插件上，无论是否进行神经保护治疗;和免疫荧光染色，以评估神经元存活率。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 594 Donkey anti-Mouse IgG secondary antibody</td>
			<td>ThermoFisher scientific</td>
			<td>A21203</td>
			<td></td>
		</tr>
		<tr>
			<td>Basal Medium Eagle (BME)</td>
			<td>ThermoFisher scientific</td>
			<td>21010046</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety cabinet Class II, Type A2</td>
			<td>NuAire</td>
			<td>NU-540-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Millipore Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Calbindin D-28K antibody (CB-955)</td>
			<td>Abcam</td>
			<td>ab82812</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>NuAire</td>
			<td>NU-5700</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar Flat Bottom 6-well Cell Culture Plates</td>
			<td>Fisher Scientific</td>
			<td>07-200-83</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips, 22 x 50 mm</td>
			<td>Fisher Scientific</td>
			<td>12-545-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Dressing forceps, straight</td>
			<td>Harvard Apparatus</td>
			<td>72-8949</td>
			<td></td>
		</tr>
		<tr>
			<td>Double edge blades</td>
			<td>Fisher Scientific</td>
			<td>50949411</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Decon Labs, Inc</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye Scissors, straight</td>
			<td>Harvard Apparatus</td>
			<td>72-8428</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Fisher Scientific</td>
			<td>16-100-127</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine 100X</td>
			<td>ThermoFisher scientific</td>
			<td>25030149</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose solution</td>
			<td>ThermoFisher scientific</td>
			<td>A2494001</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks' Balanced Salt Solution</td>
			<td>ThermoFisher scientific</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342, Trihydrochloride, Trihydrate</td>
			<td>Fisher Scientific</td>
			<td>H21492</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum, heat inactivated, New Zealand origin</td>
			<td>ThermoFisher scientific</td>
			<td>26050088</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>McIlwain Tissue Chopper</td>
			<td>Fisher Scientific</td>
			<td>NC9914528</td>
			<td></td>
		</tr>
		<tr>
			<td>Microprobes</td>
			<td>Fisher Scientific</td>
			<td>08-850</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell Cell Culture Inserts</td>
			<td>Millipore Sigma</td>
			<td>PICM0RG50</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Rapid-Flow Sterile Disposable Filter Units with PES Membrane, 250 mL</td>
			<td>ThermoFisher scientific</td>
			<td>168-0045</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1R confocal laser microscope system</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements Imaging Software</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Acros Organics</td>
			<td>41678-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20D</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>ThermoFisher scientific</td>
			<td>P10144</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating Scissors, straight</td>
			<td>Harvard Apparatus</td>
			<td>72-8403</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker Belly Dancer</td>
			<td>IBI Scientific</td>
			<td>BDRLS0003</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 8</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Dish, 60 mm w/ grip ring</td>
			<td>Fisher Scientific</td>
			<td>FB012921</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture plate, 24 well</td>
			<td>Falcon/Corning</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipettes, sterile</td>
			<td>ThermoFisher scientific</td>
			<td>13-711-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>ThermoFisher scientific</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

purkinje cell survival in organotypic cerebellar slice cultures

组织切片培养是一种强大的体外模型，它比分离的原细胞培养更密切地模拟体内条件。在产后早期发育中，已知小脑Purkinje细胞会经历一个脆弱的时期，在此期间，它们经历程序性细胞死亡。在这里，我们提供了一个详细的协议，以执行小鼠器官小脑切片培养在此关键时间。进一步标记切片，以评估Purkinje细胞存活率和神经保护治疗的有效性。这种方法对于筛选新的神经活性分子非常有价值。

如图4所示，该协议产生有机细胞小脑切片培养物，其中Purkinje细胞存活率可按照免疫荧光和图像采集步骤进行评估。Purkinje细胞被标记为抗卡宾丁D-28K（稀释1/200）和Alexa594抗小鼠（稀释1/300）抗体的组合。通过显微镜采集软件（NIS-元素）自动进行图像拼接，以获得整个小脑切片的图片。在棱镜8软件中输入了每个切片的Purkinje细胞数，以生成图表并执行统计分析。在P6，Purkinje?...

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Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

组织切片培养是研究神经发育或退行/再生过程的有力工具。在这里，我们描述了一个协议，模型在小鼠小脑切片培养中的Purkinje细胞的神经发育死亡。该方法有利于神经保护药物发现的研究。

器官细胞生存在器官细胞切片培养

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early ...

有机切片培养是研究神经发育和退行或再生过程的有力工具。这种技术可用于快速筛选候选分子的神经保护潜力。与分离的原细胞培养物相比，此方法密切模仿体内条件，因为组织集体系结构和本机细胞连接保留在各部分的平面中。在这里，我们演示了在发育中的小脑中对Purkinje细胞死亡的研究。但有机细胞切片培养同样适合在几乎每个中枢神经系统区域对神经退行性疾病进行建模。与詹妮弗·拉科托马蒙杰一起演示这个程序的将是肖恩·麦克德莫特，一个来自我实验室的技术员。在收获小脑片之前，在消毒的生物安全柜中，用一毫升真空过滤培养基填充六孔培养板的每口井，并在治疗井中加入感兴趣的药理剂，在控制井中加入同等体积的车辆。使用无菌钳子，将一个0.4微米孔径PTFE膜过滤器插入到每口井中，注意避免每个膜和介质的界面出现气泡，并在37摄氏度和5%的二氧化碳培养箱中平衡介质两个小时。要收获小脑，请使用直敷钳抓住幼崽头部的鼻子，并使用直眼剪刀将头皮从后部横向切开到中线。以同样的方式切开头骨，向外指向剪刀尖，以避免损害小脑，并使用铲子将大脑转移到含有冷HBSS和每毫升葡萄糖5毫克的60毫米盘中。使用直敷料钳仔细解剖小脑，并使用无菌弯曲细钳将组织放在组织切碎器切割台上的塑料盘上。旋转切削台以定向组织，以允许获取副石部分，并向右拉表释放旋钮以移动切削台，直到刀片位于组织的边缘。然后将切片厚度调整到 350 微米，将刀片速度调整到中等，然后启动切碎器。当整个小脑被切成片时，关闭切碎器。使用无菌钳子，将光盘放在新的 60 毫米盘上。使用转移移液器将 HBS 和葡萄糖冲洗在光盘上，使切片落入盘中。然后，尽可能少地接触样品，使用微probe分离切片。使用转移移液器选择小脑靠近 vermis 的部分到六井板中的单个细胞培养物上，并使用微插管将切片定位到每个刀片的中心。当所有切片都放置好时，小心吸进多余的HSS和葡萄糖，然后将板回细胞培养箱。对于免疫荧光染色，从每个井中去除上流液，用 PBS 清洗刀片。将切片与每台刀片的井中一毫升冷4%半甲醛和500微升在每台刀片顶部固定一小时。在固定结束时，将刀片清洗四次，10分钟，每个刀片下一毫升新鲜PBS，每次在轨道摇床上清洗每个刀片的每个刀片上洗500微升新鲜PBS。用 500 微升 PBS-TB 预填充 24 孔板的每个孔，并使用画笔将每个细胞培养中的小脑切片插入到 24 孔板的单个孔中。在室温下对切片进行渗透和阻挡一小时。在孵化结束时，在轨道摇床上，用200微升的原抗体在PBS-TB中稀释，每井过夜，温度为4摄氏度。第二天早上，在摇床上清洗4次，洗500微升新鲜PBS，然后用适当的荧光结合二次抗体在摇床的室温下将样品标记两个小时，防止光线。在孵化结束时，用每井500微升适当的核污渍对部分进行反面，在室温下10分钟，防止光线。并使用转移移液器将切片安装到玻璃幻灯片上。然后让部分完全干燥，然后用 PBS 重新水合，然后用涂有约 80 微升安装介质的盖玻片安装组织。安装介质固化后，小脑部分即可进行成像。在产后第六天，Purkinje细胞存活率低，在控制样本中，符合他们已知的脆弱性窗口。随着捐赠动物年龄的增长和离开这个关键时期，存活率会提高。用高浓度氯化钾处理小脑片，成功诱导其去极化和存活。为了获得一致和可重复的结果，选择健康的小脑部分，并尽可能高效地执行培养装置设置至关重要。培养后应用超越了免疫荧光。由于有机体切片可用于基因组蛋白质表达研究，以及使用电生理学和钙活成像监测神经元回路活动。

Research

JoVE Journal

Neuroscience

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

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 ...

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 ...

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

器官型小脑文化：细胞凋亡的挑战和检测

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

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 ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

胚胎腹侧中脑器官型片文化：一个系统来研究多巴胺能神经元发展在体外</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

浦肯野细胞树突形态的器官切片培养分析

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

嵌入后突触蛋白的免疫标记海马切片培养

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

器官切片文化研究少突胶质细胞动力学和髓鞘

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

神经血管重塑的分析Entorhino，海马脑切片培养

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

小胶质细胞的小脑颗粒细胞使用L-亮氨酸甲酯选择性耗尽

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating ...