This work was supported by a research grant MOP 119271 to JMW by the Canadian Institute of Health Research. The authors would like to thank Ms. Yao Fang Tan for her outstanding technical assistance.

注:所有动物的程序符合比较医学系的多伦多大学的动物健康和福利的指导方针。 1.准备海马脑片<ol><li>消毒使用干高压釜以下仪器在125℃:手术刀手柄（＃3）（2），标准模式钳，大（1），小解剖剪（倾斜到另一侧）（1），微勺（勺子和平坦抹刀端）（1），微抹刀（圆形和圆角锥形端）（2）中，细画笔（1），火抛光的巴斯德吸液管（2），纱布正方形，2×2英寸（5）。 </li><li>当无菌的，把工具插入一个无菌容器中，并保持盖直至使用。立即解剖前，浸在70％的乙醇溶液的仪器。 </li><li>通过加入1ml培养基/孔，并存储该板在培养箱中在35℃的开始清扫程序之前准备一个6孔培养板培养插管次5％的CO 2。 </li></ol> 2.安排解剖工具在无菌层流罩<ol><li>喷层流罩，用70％的乙醇，并从醇除去灭菌解剖仪器。允许仪表干燥而搁置在无菌培养皿，以避免醇与解剖大脑组织之间的接触。 </li><li>存款5-7毫升的2个大型无菌培养皿消毒冰冷的解剖解决方案。一盘会放松并清洁头（脏），另一种用于冷却和冲洗舀出脑（清洁）。放置无菌滤纸的培养皿盖子解剖出脑之一。 </li><li>放置一个小的，无菌滤纸的小的培养皿盖子解剖出海马中的一个。存款3-5毫升消毒冰冷的解剖解决方案2小，无菌培养皿。一盘将举办挖出海马，一会的海马部分分离时持有的部分解剖显微镜下。 </li><li>由胶带一块无菌滤纸到切割阶段和安装无菌刀片准备组织斩波。湿用无菌解剖溶液的滤纸上。 </li><li>喷雾清洁生物袋用酒精，并将其放置在层流超净工作台。 </li></ol> 3.海马解剖<ol><li>喷P7只SD大鼠幼崽，用70％的乙醇的层流净化台的外面，并迅速用层流长凳里面大无菌手术剪刀斩首动物。让头部落入冰冷解剖溶液中的培养皿1。 </li><li>在培养皿中，冲洗过的血液，并迅速转移到头部灭菌滤纸，腹侧下来。 </li><li>使用手术刀，沿背表面在矢状面切割以暴露下面的头骨。切穿皮肤，但不是底层骨，这是软的，很容易穿透大鼠吨的他的年龄。抛开这种"脏"手术刀，也不对脑组织使用。 </li><li>用小剪刀剥离（角度到另一侧）和钳，切开颅骨沿颅骨矢状缝到前囟门，在那里的冠状缝被矢状缝垂直相交的头骨解剖点。使用镊子从头骨的中线拉头骨襟翼起来了。 </li><li>放置在脑的下侧，脑干下方的微勺，轻轻抬起脑出颅骨。提起大脑暴露视神经和嗅球脑基底表面。切开这些结构用小剪刀从头骨完全分离的大脑。删除和完整的大脑转移到含有冰冷的解剖解决方案等大型培养皿。 </li><li>使用微勺，大脑转移到含有无菌滤纸的小培养皿盖。用无菌巴斯德吸管，冲洗大脑用几滴DIS的secting的解决方案，以保持组织的湿润。 </li><li>使用"干净"的手术刀切开两半球分开。转让左半球背到大培养皿微勺子上下放置半球PIA一边为后续使用冰冷的解剖解决方案。 </li><li>查看面对内侧右半球和识别边缘穹窿，白质沿内侧缘海马的一个突出带。用无菌解剖刀，使通过穹窿矢状切，但照顾因为手术刀尖端仅0.5厘米将足以切割穹窿。 </li><li>使用2微抹刀，通过将右手抹刀上脑干和起吊上覆皮质与左手刮刀移除右半球第一海马。轻轻抬起皮质揭示海马侧脑室内侧面。白色曲线，伞端，现在应该是可见的。 </li><li>抹刀的曲率对准看台伞端的自命轻轻按下伞下铲。向左滑动锅铲，沿着喙，尾轴骑，然后抬起铲背的方向，除去海马。 </li><li>海马转移到第二小培养皿用冰冷的解剖溶液。重复左侧半球相同的步骤拆下左海马。 </li><li>用微型抹刀，仔细海马转移到组织菜刀阶段。排列它们相邻并彼此平行并垂直于斩波叶片的轴线。使用画笔来定位组织并在海马的顶部加几滴解剖溶液。 </li><li>削减400微米的切片组织没有停下来删除单个片（通常他们会不会坚持到刀片）。整个海马已被切断后，用画笔来的部分轻轻地转移到第二小的培养皿剥离的解决方案。 </li><li>根据广告issecting显微镜，仔细分离使用amicro，铲和画笔浮动片。 </li><li>取出预先准备好的培养板从培养箱并置于层流罩培养插管。 </li><li>使用火抛光的巴斯德吸管，吸取4-5片放入吸管和转移切片文化插入膜的顶面。接着，调整用画笔的定位和离开的各个部分，并培养插入物的边界之间的空间。 </li><li>用无菌巴斯德吸管，去除膜的顶面多余的解剖解决方案。 注:卸下解决方案避免将组织切片放入吸管。另外，使用常规的移液管（P200或P1000）用灭菌枪头慢慢消除解决方案。 </li><li>放置在培养板用含血清的培养基中，并在海马切片放回培养箱中在35℃和5％的CO 2。 注意:如果实验通话从多个动物产生的文化，彻底清洁夹层之间的层流洁净工作台。返回仪器酒精溶液，并更换所有培养皿，滤纸和之前第二清扫消毒刀片。 </li></ol> 4.饲养和维护器官切片<ol><li>饲料文化在无菌层流洁净工作台。 </li><li>执行部分培养后2天解剖的第一馈送。使用消毒的玻璃吸管吸出旧的培养基。 </li><li>使用无菌5毫升血清吸液管来加入1 ml新鲜，无菌的，含有血清的培养基的孔中。 </li><li>轻轻地取代文化插入和照顾，以消除可能已经膜表面之下形成的气泡。 </li><li>第一进给后，介质改变每隔一天。 </li></ol> 5.孵化组织切片与胸苷类似物标签​​新生神经元<ol><li>为了探讨成熟和整合齿状颗粒细胞在海马，孵化器官切片与胸苷类似物，如溴脱氧尿苷（尿嘧啶）或Chlorodeoxyuridine（CldU）。在这里，我们使用，因为在生理盐水中的溶解度较高的CldU。 </li><li> 3DIV后，对于10微克/ ml的终浓度加入1微升10毫克/毫升CldU原液在盐水到1ml培养基。如果BrdU的被使用，校正分子量的差异的浓度。与CldU添加培养基培养孔，并培育组织与CldU含培养基2小时，在35℃。 </li><li>以下2小时温育在35℃，除去含CldU培养基并用常规给送介质替换以恢复正常喂养时间表（上面概述的）。 </li></ol> 6.组织固定和存储<ol><li>建立的时间表用于施加处理和固定组织样品开始培养实验（图2B）之前。 </li> &lt;li&gt;在预定天后解剖，准备以下在实验室通风柜:含有10-50毫升烧杯4％多聚甲醛（PFA）在磷酸盐缓冲盐水（PBS）;空烧杯丢弃培养基;小镊子;和1000毫升吸管与一次性吸头。 </li><li>去除孵化室和转移培养板（S）到通风橱。倾斜单井板镶钳。接下来，用吸管去除培养基，并转移到处置烧杯中。倾斜培养板以一定角度，以帮助确保所有培养基被除去。 </li><li>散场时间删除介质一种文化板块。当媒体已被删除，加入1 ml 4％PFA的每培养孔和密封孔板封口膜。重复许多培养板作为必要和转移板到冰箱，在4℃下放置24小时。 </li><li> 24小时后，准备在通风橱下:10-50毫升烧杯含有0.1％叠氮化钠的PBS中;空烧杯丢弃PFA（遵守安全注意事项丢弃有毒物质时）;小镊子;和吸管一次性吸头千毫升。 </li><li>按照6.4中概述用于添加煤灰的方法，但添加1ml的PBS中与叠氮化钠代替。一旦完全转移，密封孔板供将来使用通过包装边缘封口膜，并在4℃下储存在冰箱中。 </li></ol> 7.组织切片的免疫组化<ol><li>执行组织使用振动切片。以下一系列步骤有助于最大限度地从器官型培养可用组织切片的产率。 注:紧随海马解剖和切片的电镀，所述组织的厚度为约400微米。然而，2-3周，在孵育室后，组织切片会开始变平，从而产生最后一节的150-300微米厚。 </li><li>准备好以下项目第培养组织:一个＃11手术刀B拉德和处理，含冰冷PBS，微解剖钳，和一个清洁的vibratome切割阶段装入组织的玻璃培养皿。 </li><li>使用解剖刀小心沿圆形刀片膜的周边切割，以便它可以从塑料插入件壳体上卸下。离开培养切片和手术刀之间的充足的空间。 </li><li>该分离插入膜转移到含PBS的培养皿。漂洗在PBS后，用钳子向膜转移到的vibratome安装阶段。 </li><li>接下来，用手术刀来消除周围的培养切片多余膜切掉多余的材料，以创造清洁的边缘。这个步骤将确保该膜是平坦的，可以很容易地粘附在切割表面。 </li><li>放置1-2滴粘合剂上的vibratome切割阶段和散布在偶数层用22克针。散布粘合剂为矩形形状具有平行于的vibratome的切割刀片的长边。执行此步骤快，以防止干燥的粘合剂。 </li><li>使用镊子包含海马切片修整膜转移至所述切割阶段，并轻轻地定位于胶膜，并确保没有气泡。 </li><li>作为强力胶干燥，用粘膜回的PBS含培养皿转移的vibratome阶段。制备的vibratome刀片和一个48孔平板含有叠氮化钠来存储组织切片。 </li><li>使用的vibratome生成器官切片组织和转移的30微米的部分，以一个48孔板含有叠氮化钠进行存储和随后的免疫组织化学染色。 </li><li>参阅预先存在用于免疫组化染色26,29协议。 </li></ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+in+hippocampal+slice+cultures.">Neurogenesis in hippocampal slice cultures.</a> Molecular And Cellular Neurosciences. 26 (2), 241-250 (2004).</li><li>Kamada, 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=Intrinsic+and+spontaneous+neurogenesis+in+the+postnatal+slice+culture+of+rat+hippocampus.">Intrinsic and spontaneous neurogenesis in the postnatal slice culture of rat hippocampus.</a> The European Journal Of Neuroscience. 20 (10), 2499-2508 (2004).</li><li>Kim, H., Kim, E., Park, M., Lee, E., Namkoong, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+hippocampal+slice+culture+from+the+adult+mouse+brain:+a+versatile+tool+for+translational+neuropsychopharmacology.">Organotypic hippocampal slice culture from the adult mouse brain: a versatile tool for translational neuropsychopharmacology.</a> Progress in Neuro-Psychopharmacology &amp; Biological Psychiatry. 41, 36-43 (2013).</li><li>Legradi, A., Varszegi, S., Szigeti, C., Gulya, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+rat+hippocampal+slices+as+in+vitro+models+for+neurodegeneration:+Studies+on+cell+viability+and+apoptotic+processes.">Adult rat hippocampal slices as in vitro models for neurodegeneration: Studies on cell viability and apoptotic processes.</a> Brain Research Bulletin. 84 (1), 39-44 (2011).</li><li>Sadgrove, M. P., Laskowski, A., Gray, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+granule+layer+cell+count,+cell+density,+and+single-pulse+BrdU+incorporation+in+rat+organotypic+hippocampal+slice+cultures+with+respect+to+culture+medium,+septotemporal+position,+and+time+in+vitro.">Examination of granule layer cell count, cell density, and single-pulse BrdU incorporation in rat organotypic hippocampal slice cultures with respect to culture medium, septotemporal position, and time in vitro.</a> The Journal of Comparative Neurology. 497 (3), 397-415 (2006).</li><li>Mielke, J. G., 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=Cytoskeletal,+synaptic,+and+nuclear+protein+changes+associated+with+rat+interface+organotypic+hippocampal+slice+culture+development.">Cytoskeletal, synaptic, and nuclear protein changes associated with rat interface organotypic hippocampal slice culture development.</a> Developmental Brain Research. 160 (2), 275-286 (2005).</li><li>Fabian-Fine, R., Volknandt, W., Fine, A., Stewart, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+pre-+and+postsynaptic+distribution+of+AMPA+receptors+at+synapses+in+CA3+stratum+radiatum+of+hippocampal+slice+cultures+compared+with+intact+brain.">Age-dependent pre- and postsynaptic distribution of AMPA receptors at synapses in CA3 stratum radiatum of hippocampal slice cultures compared with intact brain.</a> European Journal of Neuroscience. 12 (10), 3687-3700 (2000).</li><li>Laplagne, D. A., 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=Functional+convergence+of+neurons+generated+in+the+developing+and+adult+hippocampus.">Functional convergence of neurons generated in the developing and adult hippocampus.</a> PLoS Biology. 4 (12), e409 (2006).</li><li>McDonald, H. Y., Wojtowicz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+neurogenesis+in+the+dentate+gyrus+of+adult+rats.">Dynamics of neurogenesis in the dentate gyrus of adult rats.</a> Neuroscience Letters. 385 (1), 70-75 (2005).</li><li>Stone, S. S., 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=Functional+convergence+of+developmentally+and+adult-generated+granule+cells+in+dentate+gyrus+circuits+supporting+hippocampus-dependent+memory.">Functional convergence of developmentally and adult-generated granule cells in dentate gyrus circuits supporting hippocampus-dependent memory.</a> Hippocampus. 21 (12), 1348-1362 (2011).</li><li>Wang, S., Scott, B. W., Wojtowicz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogenous+properties+of+dentate+granule+neurons+in+the+adult+rat.">Heterogenous properties of dentate granule neurons in the adult rat.</a> Journal of Neurobiology. 42 (2), 248-257 (2000).</li><li>Wojtowicz, J. M., Kee, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BrdU+assay+for+neurogenesis+in+rodents.">BrdU assay for neurogenesis in rodents.</a> Nature Protocols. 1 (3), 1399-1405 (2006).</li><li>Fritsch, R. S. E. R., Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereological+Methods,+Vol.+1:+Practical+Methods+for+Biological+Morphometry.">Stereological Methods, Vol. 1: Practical Methods for Biological Morphometry.</a> Zeitschrift f&#252;r Allgemeine Mikrobiologie. 21 (8), 630-630 (1981).</li><li>Bunk, E. C., Konig, H. G., Bonner, H. P., Kirby, B. P., Prehn, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA-induced+injury+of+mouse+organotypic+hippocampal+slice+cultures+triggers+delayed+neuroblast+proliferation+in+the+dentate+gyrus:+an+in+vitro+model+for+the+study+of+neural+precursor+cell+proliferation.">NMDA-induced injury of mouse organotypic hippocampal slice cultures triggers delayed neuroblast proliferation in the dentate gyrus: an in vitro model for the study of neural precursor cell proliferation.</a> Brain Research. 1359, 22-32 (2010).</li><li>Kolb, B., Pedersen, B., Ballermann, M., Gibb, R., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+and+postnatal+injections+of+bromodeoxyuridine+produce+age-dependent+morphological+and+behavioral+abnormalities.">Embryonic and postnatal injections of bromodeoxyuridine produce age-dependent morphological and behavioral abnormalities.</a> The Journal Of Neuroscience : The Official Journal Of The Society For Neuroscience. 19 (6), 2337-2346 (1999).</li><li>Morris, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+toxicology+of+5-bromodeoxyuridine+in+mammalian+cells.">The genetic toxicology of 5-bromodeoxyuridine in mammalian cells.</a> Mutation Research. 258 (2), 161-188 (1991).</li><li>Bannigan, J., Langman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellular+effect+of+5-bromodeoxyuridine+on+the+mammalian+embryo.">The cellular effect of 5-bromodeoxyuridine on the mammalian embryo.</a> Journal Of Embryology And Experimental Morphology. 50, 123-135 (1979).</li><li>Breunig, J. J., Arellano, J. I., Macklis, J. D., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Everything+that+glitters+isn't+gold:+a+critical+review+of+postnatal+neural+precursor+analyses.">Everything that glitters isn't gold: a critical review of postnatal neural precursor analyses.</a> Cell Stem Cell. 1 (6), 612-627 (2007).</li><li>Duque, A., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+effects+of+bromodeoxyuridine+and+[3H]thymidine+incorporation+into+DNA+on+cell+proliferation,+position,+and+fate.">Different effects of bromodeoxyuridine and [3H]thymidine incorporation into DNA on cell proliferation, position, and fate.</a> The Journal Of Neuroscience : The Official Journal Of The Society For Neuroscience. 31 (42), 15205-15217 (2011).</li><li>Namba, T., Mochizuki, H., Onodera, M., Namiki, H., Seki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+neurogenesis+in+hippocampal+slice+cultures:+early+in+vitro+labeling+of+neural+precursor+cells+leads+to+efficient+neuronal+production.">Postnatal neurogenesis in hippocampal slice cultures: early in vitro labeling of neural precursor cells leads to efficient neuronal production.</a> Journal of Neuroscience Research. 85 (8), 1704-1712 (2007).</li></ol>

The authors declare that they have no competing financial interests.

以下CldU（或尿嘧啶）施用，施用药剂的时间轴可以被选择为目标时特定发育窗户新生儿DGCS。例如，一个假想的代理可以在第二星期后CldU注射，这是建议用未成熟的神经元是在发育阶段的GABA被去极化的年龄相一致时被应用。使用该协议的将来的研究可以适应的药理剂和暴露于"量体裁衣"的方法对感兴趣的具体的实验问题的窗口。 用于确定切片文化是产后神经研究的一个有效?...

Adult neurogenesis in the mammalian hippocampus represents a remarkable example of the brain&#8217;s innate capacity for adaptability and plasticity. Dentate granule cells (DGCs) are generated from a renewable pool of neural progenitor cells in the hippocampal dentate gyrus, which is one of the two presently well-characterized neurogenic regions in the mammalian brain, and is thought to be particularly important for learning and memory. The hippocampus is part of the limbic system and has a deep location within the mammalian brain, which makes it a difficult target for precise pharmacological manipulation. Additionally, aberrant neurogenesis has been implicated in conditions, such as epilepsy, schizophrenia, and Alzheimer&#8217;s disease, which has prompted interest in understanding the influence of various pharmacological agents during the maturation and survival of newborn neurons. The distinction between postnatal and adult neurogenesis is blurred and previous studies have shown that many features of in vivo neuronal development in the early postnatal period and adulthood are similar25. Here we emphasize postnatal neurogenesis and suggest possible applications to adult neurogenesis.
Organotypic slice cultures provide an efficient in vitro method for studying various physiological properties of the mammalian hippocampus. The value of slice cultures prepared from rodent brains can be summarized in three main qualities: 1) the protocol is straightforward and requires readily available materials; 2) slice cultures allow for pharmacological studies that eliminate complex variables such as the deep anatomic location of the hippocampus and circumvents the blood brain barrier1; and 3) the well characterized structure of the hippocampus and tri-synaptic circuit is preserved2. Previous investigators have used the organotypic hippocampal culture to study synaptic development and physiology3,4, gliogenesis5-7, ischemic brain damage8,9, neuroprotection and neurorepair10-12 as well as epilepsy13-15.The slice cultures could also provide a useful model system allowing for the monitoring of cell development in conjunction with labeling of cells with green fluorescent protein (GFP) or other vital markers.
Slice cultures have also been previously employed to study postnatal hippocampal neurogenesis16-19, but one important factor in the majority of these studies is the well-characterized degeneration that results from explanting tissue from adult animals after approximately 2 weeks in vitro20,21. For this reason, slice cultures are typically prepared from early post-natal (P5-P10) mice or rat pups, which utilizes the improved viability of early postnatal brain tissue for culturing22. While previous studies have shown that the early postnatal and adult hippocampus differ with regards to synaptic physiology and the expression of specific neuronal subtypes23,24, there is substantial conservation of the choreographed developmental program that newborn dentate granule cells proceed through during maturation25. Additionally, recent studies have suggested that the physiological characteristics of newborn DGCs in culture are very similar to immature neurons in the acute hippocampal slice preparation16.

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			<td height="46" rowspan="2" style="height:47px;width:265px;">Name of Reagent/ Equipment</td>
			<td rowspan="2" style="width:131px;">Company</td>
			<td rowspan="2" style="width:135px;">Catalog Number</td>
			<td rowspan="2" style="width:239px;">Comments/Description</td>
		</tr>
		<tr height="20">
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:265px;">5-chloro-2&#39;-deoxyuridine (CldU)</td>
			<td style="width:131px;">MP Biomedicals</td>
			<td align="right" style="width:135px;">105478</td>
			<td style="width:239px;">Hazardous, Carcinogenic</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:265px;">Cell culture inserts, 30mm diameter, 0.4&#181;m pore size</td>
			<td style="width:131px;">Thermo scientific&#160;</td>
			<td align="right" style="width:135px;">140660</td>
			<td style="width:239px;">Nuclon delta coating on these inserts provides better tissue adhesion and improves slice quality.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Conical Centrifuge tubes (sterile)</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:135px;">14-432-22</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Dissector scissors (angled to side)</td>
			<td style="width:131px;">Fine Science Tools&#160;</td>
			<td style="width:135px;">14082-09</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:265px;">Minimum essential medium (MEM)</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:135px;">11095; liquid</td>
			<td style="width:239px;">Store at 4&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Eclipse Ni-U fluorescent microscope</td>
			<td style="width:131px;">Nikon</td>
			<td style="width:135px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:265px;">Glue for tissue</td>
			<td style="width:131px;">Krazy Glue</td>
			<td style="width:135px;">KG585</td>
			<td style="width:239px;">Use minimum amount of glue to achieve adhesion as any tissue exposed to glue will be unusable for IHC.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Hank&#8217;s Balanced Salt Solution (HBSS) (500 mL)</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:135px;">14025-092</td>
			<td style="width:239px;">Store at 4&#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Horse Serum Heat Inactivated (500 mL)</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:135px;">16050-122</td>
			<td style="width:239px;">Make 50 mL aliquots and store at -20&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:265px;">Kimwipes</td>
			<td style="width:131px;">Kimberly-Clarke</td>
			<td style="width:135px;">TW 31KYPBX</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:265px;">Modified glass pipettes (bottom of Pasteur pipette removed and edge smoothed with Bunsen flame)</td>
			<td style="width:131px;"></td>
			<td style="width:135px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Petri Dish (100mm x 15mm) and (60mm x 15mm)</td>
			<td style="width:131px;">Fisher Brand</td>
			<td style="width:135px;">FB0875712 and FB0875713A</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Scalpel blades #11</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:135px;">10011-00</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Scalpel handle #3</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:135px;">10003-12</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Serological Pipettes</td>
			<td style="width:131px;">Sorfa Medical Plastic Co.</td>
			<td style="width:135px;">P8050</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Standard Pattern forceps</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:135px;">11000-12</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Sterile vacuum filter</td>
			<td style="width:131px;">Thermo-Scientific</td>
			<td style="width:135px;">565-0020</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Surgical Scissors</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:135px;">14054-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Syringe driven filter unit</td>
			<td style="width:131px;">Millipore-Millex</td>
			<td style="width:135px;">SLGP033RS</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Tissue chopper with moveable stage</td>
			<td style="width:131px;">Stoelting&#160;</td>
			<td align="right" style="width:135px;">51425</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:265px;">Fine tip paintbrush</td>
			<td style="width:131px;"></td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

organotypic slice cultures for studies of postnatal neurogenesis

在这里，我们描述了使用器官切片培养技术研究海马神经发生产后的啮齿类动物的大脑的技术。该方法保持了海马的特征地形的形态，同时允许直接应用的药剂给显影海马齿状回。此外，切片培养物可维持4周，因此，允许一个研究新生儿颗粒神经元的成熟过程。切片培养允许有效药理操纵海马切片的同时排除复杂的变量，例如与海马的深解剖位置，以及血脑屏障的不确定性。由于这些原因，我们试图专门优化器官切片文化为产后神经研究。

确定是否器官培养物将适合于成年神经研究需要它们满足两个主要标准:1），该片段保持海马切片的特性形态学特征后10-21天内在体外 （DIV），和2），初生DGCS可以量化采用常用于成年神经研究标准的免疫组化技术。关于第一个标准， 图1A和1B突出保存海马形态。特征，如齿状回（DG），CA1和CA3地区是容易辨认。 对于第二准则， 图1C（</stro...

Watch this Scientific Journal Video about Organotypic Slice Cultures for Studies of Postnatal Neurogenesis at JoVE.com

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

在这里，我们描述了学习的海马神经发生产后使用的器官切片培养技术的技术。此方法允许在体外操纵成年神经发生，并允许直接应用的药剂的培养海马。

器官切片文化为产后神经再生的研究

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

organotypic-slice-cultures-for-studies-of-postnatal-neurogenesis

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Developmental Biology

12.2K 次观看.  University of Toronto. 在这里，我们描述了学习的海马神经发生产后使用的器官切片培养技术的技术。此方法允许在体外操纵成年神经发生，并允许直接应用的药剂的培养海马。 

视频: Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

小鼠海马组织的前子宫内电和器官切片文化

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

科研

发育生物学

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

转基因器官皮肤培养使用灭活人真皮代

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell lines. However, primary cell cultures usually have a finite life span and need to be frequently re-established. Fibroblasts are an easily accessible source of primary cells. Here, we discuss a simple and quick experimental procedure to establish primary fibroblast cultures from ears and tails of mice. The protocol can be used to establish primary fibroblast cultures from ears stored at RT for up to 10 days. When the protocol is carefully followed, contaminations are unlikely to occur despite the use of non-sterile tissue stored for extended time in some cases. Fibroblasts proliferate rapidly in culture and can be expanded to substantial numbers before undergoing replicative senescence.

We describe a simple and quick experimental procedure for generating primary fibroblasts from the ears and tails of mice. The procedure does not require special animal training and can be used for the generation of fibroblast cultures from ears stored at RT for up to 10 days.

从鼠耳和尾组织生成主成纤维细胞培养

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell ...

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-CreERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects.

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

成像清除胚胎和出生后心脏在单细胞分辨率

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

细胞周期变化的可视化和核测定心肌出生后

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate embryonic origins and functions &#8212; the neurohypophysis and the adenohypophysis. The developing mouse pituitary gland is tiny and delicate with an elongated oval shape. A coronal section is preferred to display both the adenohypophysis and neurohypophysis in a single slice of the mouse pituitary.
The goal of this protocol is to achieve proper pituitary coronal sections with well-preserved tissue architectures from developing mice. In this protocol, we describe in detail how to dissect and process pituitary glands properly from developing mice. First, mice are fixed by transcardial perfusion of formaldehyde prior to dissection. Then three different dissecting techniques are applied to obtain intact pituitary glands depending on the age of mice. For fetal mice aged embryonic days (E) 17.5 - 18.5 and neonates up to 4 days, the entire sella regions including the sphenoid bone, gland, and trigeminal nerves are dissected. For pups aged postnatal days (P) 5 - 14, the pituitary glands connected with trigeminal nerves are dissected as a whole. For mice over 3 weeks old, the pituitary glands are carefully dissected free from the surrounding tissues. We also display how to embed the pituitary glands in a proper orientation by using the surrounding tissues as landmarks to obtain satisfying coronal sections. These methods are useful in analyzing histological and developmental features of pituitary glands in developing mice.

We present a protocol to dissect pituitary glands and prepare pituitary coronal sections from developing mice.

发育小鼠垂体的解剖和冠状切片制备

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

三维脊髓前庭和听觉感官器官的培养

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

小鼠胰腺内分泌细胞的单细胞 Transcriptomic 分析

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Neurogenesis Using P19 Embryonal Carcinoma Cells

The P19 cell line derived from a mouse embryo-derived teratocarcinoma has the ability to differentiate into the three germ layers. In the presence of retinoic acid (RA), the suspension cultured P19 cell line is induced to differentiate into neurons. This phenomenon is extensively investigated as a neurogenesis model in vitro. Therefore, the P19 cell line is very useful for molecular and cellular studies associated with neurogenesis. However, protocols for neuronal differentiation of P19 cell line described in the literature are very complex. The method developed in this study are simple and will play a part in elucidating the molecular mechanisms in neurodevelopmental abnormalities and neurodegenerative diseases.

The P19 mouse embryonic carcinoma cell line (P19 cell line) is widely used for studying the molecular mechanism of neurogenesis with great simplification compared to in vivo analysis. Here, we present a protocol for retinoic acid-induced neurogenesis in the P19 cell line.

使用P19胚胎癌细胞的神经发生

The P19 cell line derived from a mouse embryo-derived teratocarcinoma has the ability to differentiate into the three germ layers. In the presence of ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

毛因神经发生期间诺奇信号动力学的实时生物发光成像

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...