Name: live imaging of drosophila larval neuroblasts
Uploaded: 2014-07-07T15:00:00
Description: Watch this Scientific Journal Video about Live Imaging of Drosophila Larval Neuroblasts at JoVE.com

这项工作是由校内的研究在健康/ NHLBI（1ZIAHL006126）和Lenfant生物医学博士后奖学金的颁发给DAL民族院校划分的支持。

1，所需材料的制备植三龄幼虫脑<ol><li>准备所需的显微切割工具。 <ol><li>锐意2解剖工具（手术刀和钩），首先将单个解剖针向每个引脚架。用手或钳（ 图1C）固定销紧密。 </li><li>使用一对老镊子弯曲1解剖针到约120度角。为此在解剖显微镜下，以确保销的上半部分平放时，该引脚保持器在手持。该工具将被用作手术刀按住或切片穿过组织。 </li><li>使用一对老钳子弯曲第二销到将被?...

<ol>
	<li>Truman, J. W., Bate, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+patterns+of+neurogenesis+in+the+central+nervous+system+of+Drosophila+melanogaster.">Spatial and temporal patterns of neurogenesis in the central nervous system of Drosophila melanogaster.</a> Developmental Biology. 125, 145-157 (1988).</li><li>Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+markers+for+identified+neuroblasts+and+ganglion+mother+cells+in+the+Drosophila+central+nervous+system.">Molecular markers for identified neuroblasts and ganglion mother cells in the Drosophila central nervous system.</a> Development. 116, 855-863 (1992).</li><li>Lerit, D. A., Smyth, J. T., Rusan, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle+asymmetry+for+proper+fitness,+function,+and+fate.">Organelle asymmetry for proper fitness, function, and fate.</a> Chromosome research. 21, 271-286 (2013).</li><li>Bello, B. C., Izergina, N., Caussinus, E., Reichert, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplification+of+neural+stem+cell+proliferation+by+intermediate+progenitor+cells+in+Drosophila+brain+development.">Amplification of neural stem cell proliferation by intermediate progenitor cells in Drosophila brain development.</a> Neural development. 3, 5 (2008).</li><li>Bowman, S. K., 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=The+tumor+suppressors+Brat+and+Numb+regulate+transit-amplifying+neuroblast+lineages+in+Drosophila.">The tumor suppressors Brat and Numb regulate transit-amplifying neuroblast lineages in Drosophila.</a> Developmental Cell. 14, 535-546 (2008).</li><li>Boone, J. Q., Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Drosophila+type+II+neuroblast+lineages+containing+transit+amplifying+ganglion+mother+cells.">Identification of Drosophila type II neuroblast lineages containing transit amplifying ganglion mother cells.</a> Dev Neurobiol. 68, 1185-1195 (2008).</li><li>Homem, C. C., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+neuroblasts:+a+model+for+stem+cell+biology.">Drosophila neuroblasts: a model for stem cell biology.</a> Development. 139, 4297-4310 (2012).</li><li>Savoian, M. S., Rieder, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitosis+in+primary+cultures+of+Drosophila+melanogaster+larval+neuroblasts.">Mitosis in primary cultures of Drosophila melanogaster larval neuroblasts.</a> Journal of Cell Science. 115, 3061-3072 (2002).</li><li>Fleming, S. L., Rieder, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flattening+Drosophila+cells+for+high-resolution+light+microscopic+studies+of+mitosis+in+vitro.">Flattening Drosophila cells for high-resolution light microscopic studies of mitosis in vitro.</a> Cell Motil Cytoskeleton. 56, 141-146 (2003).</li><li>Buffin, E., Lefebvre, C., Huang, J., Gagou, M. E., Karess, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruitment+of+Mad2+to+the+kinetochore+requires+the+Rod/Zw10+complex.">Recruitment of Mad2 to the kinetochore requires the Rod/Zw10 complex.</a> Current biology. 15, 856-861 (2005).</li><li>Basto, R., 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=Flies+without+centrioles.">Flies without centrioles.</a> Cell. 125, 1375-1386 (2006).</li><li>Lucas, E. P., Raff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintaining+the+proper+connection+between+the+centrioles+and+the+pericentriolar+matrix+requires+Drosophila+centrosomin.">Maintaining the proper connection between the centrioles and the pericentriolar matrix requires Drosophila centrosomin.</a> J. Cell Biol. 178, 725-732 (2007).</li><li>Basto, R., 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=Centrosome+amplification+can+initiate+tumorigenesis+in+flies.">Centrosome amplification can initiate tumorigenesis in flies.</a> Cell. 133, 1032-1042 (2008).</li><li>Conduit, P. T., Raff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cnn+dynamics+drive+centrosome+size+asymmetry+to+ensure+daughter+centriole+retention+in+Drosophila+neuroblasts.">Cnn dynamics drive centrosome size asymmetry to ensure daughter centriole retention in Drosophila neuroblasts.</a> Curr. Biol. 20, 2187-2192 (2010).</li><li>Forer, A., Pickett-Heaps, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochalasin+D+and+latrunculin+affect+chromosome+behaviour+during+meiosis+in+crane-fly+spermatocytes.">Cytochalasin D and latrunculin affect chromosome behaviour during meiosis in crane-fly spermatocytes.</a> Chromosome research. 6, 533-549 (1998).</li><li>Rebollo, 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=Functionally+unequal+centrosomes+drive+spindle+orientation+in+asymmetrically+dividing+Drosophila+neural+stem+cells.">Functionally unequal centrosomes drive spindle orientation in asymmetrically dividing Drosophila neural stem cells.</a> Dev. Cell. 12, 467-474 (2007).</li><li>Januschke, J., Llamazares, S., Reina, J., Gonzalez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+neuroblasts+retain+the+daughter+centrosome.">Drosophila neuroblasts retain the daughter centrosome.</a> Nat. Commun. 2, 243 (2011).</li><li>Januschke, 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=Centrobin+controls+mother-daughter+centriole+asymmetry+in+Drosophila+neuroblasts.">Centrobin controls mother-daughter centriole asymmetry in Drosophila neuroblasts.</a> Nat Cell Biol. 15, 241-248 (2013).</li><li>Homem, C. C., Reichardt, I., Berger, C., Lendl, T., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-Term+Live+Cell+Imaging+and+Automated+4D+Analysis+of+Drosophila+Neuroblast+Lineages.">Long-Term Live Cell Imaging and Automated 4D Analysis of Drosophila Neuroblast Lineages.</a> PLoS One. 8, (2013).</li><li>Neumuller, R. 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=Genome-wide+analysis+of+self-renewal+in+Drosophila+neural+stem+cells+by+transgenic+RNAi.">Genome-wide analysis of self-renewal in Drosophila neural stem cells by transgenic RNAi.</a> Cell Stem Cell. 8, 580-593 (2011).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Siegrist, S. E., Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrinsic+cues+orient+the+cell+division+axis+in+Drosophila+embryonic+neuroblasts.">Extrinsic cues orient the cell division axis in Drosophila embryonic neuroblasts.</a> Development. 133, 529-536 (2006).</li><li>Broadus, J., Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrinsic+cues,+intrinsic+cues+and+microfilaments+regulate+asymmetric+protein+localization+in+Drosophila+neuroblasts.">Extrinsic cues, intrinsic cues and microfilaments regulate asymmetric protein localization in Drosophila neuroblasts.</a> Curr. Biol. 7, 827-835 (1997).</li><li>Siller, K. H., Serr, M., Steward, R., Hays, T. S., Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+Drosophila+brain+neuroblasts+reveals+a+role+for+Lis1/dynactin+in+spindle+assembly+and+mitotic+checkpoint+control.">Live imaging of Drosophila brain neuroblasts reveals a role for Lis1/dynactin in spindle assembly and mitotic checkpoint control.</a> Molecular biology of the cell. 16, 5127-5140 (2005).</li><li>Britton, J. S., Edgar, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+control+of+the+cell+cycle+in+Drosophila:+nutrition+activates+mitotic+and+endoreplicative+cells+by+distinct+mechanisms.">Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms.</a> Development. 125, 2149-2158 (1998).</li><li>Siller, K. H., Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lis1/dynactin+regulates+metaphase+spindle+orientation+in+Drosophila+neuroblasts.">Lis1/dynactin regulates metaphase spindle orientation in Drosophila neuroblasts.</a> Developmental Biology. 319, 1-9 (2008).</li><li>Cabernard, C., Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apical/basal+spindle+orientation+is+required+for+neuroblast+homeostasis+and+neuronal+differentiation+in+Drosophila.">Apical/basal spindle orientation is required for neuroblast homeostasis and neuronal differentiation in Drosophila.</a> Dev. Cell. 17, 134-141 (2009).</li><li>Januschke, J., Gonzalez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interphase+microtubule+aster+is+a+determinant+of+asymmetric+division+orientation+in+Drosophila+neuroblasts.">The interphase microtubule aster is a determinant of asymmetric division orientation in Drosophila neuroblasts.</a> J. Cell Biol. 188, 693-706 (2010).</li><li>Cabernard, C., Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+neuroblast+lineages+within+intact+larval+brains+in+Drosophila.">Live imaging of neuroblast lineages within intact larval brains in Drosophila.</a> Cold Spring Harb Protoc. 2013, 970-977 (2013).</li><li>Rusan, N. M., Akong, K., Peifer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Putting+the+model+to+the+test:+are+APC+proteins+essential+for+neuronal+polarity,+axon+outgrowth,+and+axon+targeting.">Putting the model to the test: are APC proteins essential for neuronal polarity, axon outgrowth, and axon targeting.</a> J. Cell Biol. 183, 203-212 (2008).</li><li>Lerit, D. A., Rusan, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PLP+inhibits+the+activity+of+interphase+centrosomes+to+ensure+their+proper+segregation+in+stem+cells.">PLP inhibits the activity of interphase centrosomes to ensure their proper segregation in stem cells.</a> J. Cell Biol. 202, 1013-1022 (2013).</li><li>Morris, L. X., Spradling, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+live+imaging+provides+new+insight+into+stem+cell+regulation+and+germline-soma+coordination+in+the+Drosophila+ovary.">Long-term live imaging provides new insight into stem cell regulation and germline-soma coordination in the Drosophila ovary.</a> Development. 138, 2207-2215 (2011).</li><li>Prasad, M., Jang, A. C., Starz-Gaiano, M., Melani, M., Montell, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+culturing+Drosophila+melanogaster+stage+9+egg+chambers+for+live+imaging.">A protocol for culturing Drosophila melanogaster stage 9 egg chambers for live imaging.</a> Nat. Protoc. 2, 2467-2473 (2007).</li><li>Rusan, N. M., Peifer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+a+novel+centrosome+cycle+in+asymmetric+cell+division.">A role for a novel centrosome cycle in asymmetric cell division.</a> J. Cell Biol. 177, 13-20 (2007).</li><li>Martinez-Campos, M., Basto, R., Baker, J., Kernan, M., Raff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+pericentrin-like+protein+is+essential+for+cilia/flagella+function,+but+appears+to+be+dispensable+for+mitosis.">The Drosophila pericentrin-like protein is essential for cilia/flagella function, but appears to be dispensable for mitosis.</a> J. Cell Biol. 165, 673-683 (2004).</li><li>Dix, C. I., Raff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Spd-2+recruits+PCM+to+the+sperm+centriole,+but+is+dispensable+for+centriole+duplication.">Drosophila Spd-2 recruits PCM to the sperm centriole, but is dispensable for centriole duplication.</a> Curr. Biol. 17, 1759-1764 (2007).</li><li>Moutinho-Santos, T., Sampaio, P., Amorim, I., Costa, M., Sunkel, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+localisation+of+the+mitotic+POLO+kinase+shows+a+highly+dynamic+association+with+the+mitotic+apparatus+during+early+embryogenesis+in+Drosophila.">In vivo localisation of the mitotic POLO kinase shows a highly dynamic association with the mitotic apparatus during early embryogenesis in Drosophila.</a> Biol. Cell. 91, 585-596 (1999).</li><li>Megraw, T. L., Kilaru, S., Turner, F. R., Kaufman, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+centrosome+is+a+dynamic+structure+that+ejects+PCM+flares.">The centrosome is a dynamic structure that ejects PCM flares.</a> J. Cell Sci. 115, 4707-4718 (2002).</li></ol>

活细胞成像是用来研究，规范ACD干细胞，细胞谱系的分化机制的宝贵方法，以及复杂的组织形态。虽然研究组历来采用了各种技术来可视化NB ACD，这些方法可大致分为两类即主要与脑组织是否是原封不动或解离的差异。 成像分离的NB有其优势。为1，解离的NB更易图像，因为它们是特别容易识别作为最大的（直径约12微米）的细胞在培养皿。此外，几乎所有的干细胞，培养在玻?...

干细胞保持分化的平衡和自我更新的早期发展过程中产生细胞多样性和替代受损的细胞在成体组织。这种稳态的调节可以防止两者的损耗和过度膨胀的干细胞群体，这可能会导致有害的组织变性或肿瘤的发生。 神经母细胞（NBS）是果蝇中枢神经系统（ 图1A）在胚胎阶段1，2是第一种形式的广泛研究的神经干细胞。 NB的经过反复多轮的不对称细胞分裂（ACD），产生不等价的两个注定细胞：一个自我更新的干细胞和分化细胞。 ACD是由中心体，充当大多数细胞3的微管组织中心的非膜性细胞器引导。在有丝分裂，NB中心体组织和定向双极纺锤体沿顶 - 基底极性性轴。待分割NB的乳沟，根尖命运决定了指定干细胞的命运，和基底命运决定指定分化，被分隔成不平等的子细胞。 在幼虫中央脑，两种类型的NB可以通过它们的数量，位置，转录因子的表达，细胞谱系（ 图1A）4-6相区别。 I型的NB是最丰富的，并且其中约90填充大脑7的各视叶的前部和后部两侧。这些NB的表达的转录因子Asense（ASE），并且它们特征性地划分成一个自我更新的NB和1小神经节母细胞（GMC; 图1B）。每个GMC经历了一个单一的终端部门产生两个神经元或神经胶质细胞（ 图1B）。与此相反，8型II的NB是填充每个视叶的后侧缺乏日月光表达式5。他们经过ACD要生成一个自我更新的NB和一个中间神经祖（INP）。 该INP，反过来，划分不对称的三到五倍。所有这些分歧导致了INP的再生和生产单GMC 4。统，具体的NB身份和GMC出生的时间次序引起的成人中枢神经系统的神经细胞的惊人的多样性。 了解细胞生物学的基础是NB ACD已通过使用活细胞成像技术大大提高。研究人员使用到的图像实时的NB发布的协议有很大的不同。但是，总的来说，这些方法可以被分成由幼虫大脑是否保持不变或机械分离区分两种主要类别。这两种方法具有不同的优点和缺点取决于研究者的应用。 早期的报告揭示活NB细胞分裂本期开始涉及一定程度的幼虫大脑手动解离。这些协议的细节涂抹8或戏弄9外脑，以发展国家统计局对玻璃盖玻片短期原代培养。以改善成像，该圆的NB通常是扁平的上与任一载玻片8或琼脂糖垫9的盖玻片。虽然扁平的细胞具有改进的光学系统，这些技术往往导致NB的有丝分裂缺陷，包括卵裂沟的回归和无法分裂一次以上。因此，包含手工解离和幼虫脑组织的物理畸变的协议一般只适用于非常短期的（ 即 ，，一个细胞周期或更少）的应用程序。同样地，半的挤压或完全压扁玻璃载玻片和盖玻片之间完整脑限制一个时间可能会花费成像一个给定的样本，以一个约30分钟的时间内10。尽管有此项限制，氏的方法已经成功地11-14使用。 最近努力图像实时解离的NB极限的分离细胞的物理变形。这些技术的应用中，有必要维持细胞培养物中对多个细胞分裂周期中特别有用。例如，分散的细胞从手动分离大脑可以部分地埋入在由纤维蛋白原和凝血酶15长期成像16的混合物制成的凝块。可替代地，外植大脑可能是第一化学解离与酶胶原酶，通过移液管尖下一个手动打乱重复通道，并且随后接种于聚-L-赖氨酸包被的玻璃底培养皿17，18。镀膜玻璃的菜肴与任何纤维蛋白原或聚-L-赖氨酸促进附件和轻微的圆形细胞的扩散，使它们尽可能靠近盖玻片尽量不重大的物理变形。在additi上，这些方法涉及可方便地更换药理扰动实验18培养基中培养国家统计局。总体而言，直接电镀分散的NB改善成像光学元件在不牺牲长期的成像能力。近日，游离描述国家统计局的长期培养的协议已经有详细19。 然而，这种方法并非没有其局限性。有几个注意事项，以实时成像分离的NB。在分散的细胞的一个字段，例如，它是很难识别在空间组织中的完整的脑1特定NB谱系。同样，区分类型I与类型分离的组织内II的NB也是具有挑战性的无谱系示踪物或亚型特异性转基因，如worniu-GAL4，asense-GAL80 20的表达。虽然这些转基因是在一些应用中非常有用，它们限制了研究人员对这些转基因前在UAS增强子元件21的控制下，按下并需要更复杂的遗传计划。的研究，涉及的NB的空间或时间的发展，因此，需要在一个完整的组织的情况下体内成像。 此外，分离的胚胎的NB的研究表明，对于关键的极性相间的定位决定因素22，23相邻的小区的物理接触是至关重要的。这些研究表明完全隔离的NB不再保持不变有丝分裂纺锤体轴。相反，这些细胞显示出更多的随机主轴和连续GMC芽之间的分离，宽角度或许是由于根尖定位中心体22的损失。虽然幼虫国家统计局和他们相邻小区之间的关系还没有被广泛研究，这是值得考虑的幼虫干细胞微环境可能扑来细胞极性或其他行为。为了保持幼虫的NB的生理情况下，我们形象的ACD从整个大脑。 鉴于与成像分离的NB相关的固有局限性，我们的实验室和其他人建立了协议，从整个幼虫的大脑图像连续两轮NB的ACD。技术，以图像完整脑常与柔性膜，以防止组织变形或损坏，并允许气体交换取代玻璃的刚性表面上的培养容器的一侧。一些方法涉及装配取出的大脑中的昆虫培养用培养基，血清和抗坏血酸与10幼虫24中分离的脂肪体的混合物。分离出的脂肪组织被添加，因为它们分泌可以刺激NB细胞分裂25有丝分裂原。这个协议保留组织的完整性超过3小时，因此，适合于长期成像24，26-28。近日，描述LON协议在抗坏血酸，血清和脂肪组织的存在完好幼虫大脑的克长期成像已详述29。重要的是，因为该组织既不暴露也不固定，这种方式是为应用而培养介质被交换的有用（ 例如 ，药物研究，免疫耗竭， 等等 ）。 我们的实验室简化了整个安装现场准备幼虫大脑长期成像30，31。主要的优点我们的通讯协议在别人就是它的简单：我们的观察表明既不血清，抗坏血酸，胰岛素，也不胖的身体是必要的添加剂，以图像连续两轮NB的ACD。虽然添加剂，如胰岛素，已可用于干细胞分裂32和果蝇卵巢33集体细胞迁移的长时间成像，它们似乎是可有可无的NB ACD，作为我们的成像介质包括STA的简单混合物的ndard昆虫细胞培养用培养基补充有抗生素 - 抗真菌剂以防止污染。通过使用可重复使用的透气性或玻璃底培养皿中，我们已经能够维持几轮连续NB的ACD的。相同的外植样品可以容易地被用于免疫和其他程序与固定的组织。在这个协议中，我们详细介绍完整的幼虫大脑的解剖，以及编制大脑的实时和固定的分析。

<table border="0" cellpadding="0" cellspacing="0" width="851">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Lumox Tissue Culture Dish 50X12 mm</td>
			<td style="width:185px;">Sarstedt</td>
			<td style="width:119px;">94.6077.410</td>
			<td style="width:167px;">A reusable culture dish with a gas-permeable and optically clear film base.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Schneider&#39;s S2 Medium</td>
			<td style="width:185px;">Invitrogen</td>
			<td style="width:119px;">21720-024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Gibco Antibiotic-Antimycotic (100x)</td>
			<td style="width:185px;">Invitrogen</td>
			<td style="width:119px;">15240-062</td>
			<td>A supplement containing penicillin (10,000 U/mL), streptomycin (10,000 mg/mL), and amphotericin B (25 mg/mL) that is added to Schneider&#39;s S2 media to prevent bacterial and fungal contamination.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Glass coverslips, #1.5 22X22 mm</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:119px;">12-541-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Halocarbon oil 700</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:119px;">H8898</td>
			<td>A high viscosity inert oil that is clear and colorless.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">pair of nickel-plated pin holders, 17 cm long</td>
			<td style="width:185px;">Fine Science Tools</td>
			<td style="width:119px;">26018-17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Minutien dissecting pins</td>
			<td style="width:185px;">Fine Science Tools</td>
			<td style="width:119px;">26002-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">pair of Dumont #5 Forceps</td>
			<td style="width:185px;">Fine Science Tools</td>
			<td style="width:119px;">11252-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Dissecting needle/probe with plastic handle</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:119px;">08-965-A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Syringe filter, 0.22 mm pore size</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:119px;">09-719A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Syringe 5 mL&#160;</td>
			<td style="width:185px;">BD Biosciences</td>
			<td style="width:119px;">309646</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">plastic transfer pipet</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:119px;">S30467-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:380px;">paraformaldehyde, 32% EM-grade</td>
			<td style="width:185px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15714</td>
			<td>CAUTION: Formaldehyde is toxic; it should be handled in a fume hood. Follow MSDS guidelines for safe handling.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">DAPI (4&#39;, 6-diamidino-2-phenylindole, dihydrochloride)</td>
			<td style="width:185px;">Invitrogen</td>
			<td style="width:119px;">D1306</td>
			<td>A dye that labels DNA and is excited by UV light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Aqua/PolyMount; Polysciences, Inc.</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:119px;">18606-20</td>
			<td>A water-soluble mounting medium.</td>
		</tr>
	</tbody>
</table>

live imaging of drosophila larval neuroblasts

干细胞不对称分裂产生两个子代细胞与不平等的命运潜能：是一个自我更新的干细胞和分化细胞。鉴于其相关性发育和疾病，了解支配不对称的干细胞分裂一直是研究的一个强大的区域机制。因为他们是基因听话，并进行连续几轮细胞分裂约每小时一次， 果蝇中枢神经系统，或神经母细胞的干细胞，是不可缺少的型号为干细胞分裂的研究。约100个​​神经干细胞是位于每两个幼虫脑叶的表面附近，使该模型系统，用于实时成像显微镜研究特别有用。在这项工作中，我们将回顾一些方法广泛用于可视化的干细胞分裂，和我们处理的相对优势和那些采用分离与完整的大脑组织中的技术缺点。我们还详细介绍了simplifIED协议，用于从三龄幼虫活细胞成像和固定分析应用外植整个大脑。

实时成像已经阐明了多项调控NB的亚洲合作对话机制。前图像的采集，它是必要的，以确定最佳成像条件。有必要与活细胞成像时间平衡帧捕获率。对细胞的分析，例如，增加的时间和可能需要的光学分辨率。当可视化单一NB细胞周期（ 图4A），在该图像被捕获的速率可以增加，而不会引入光损伤。通常情况下，单个细胞分裂事件可以在约10-30秒的时间间隔进行监测。以提高时间分辨?...

Watch this Scientific Journal Video about Live Imaging of Drosophila Larval Neuroblasts at JoVE.com

Live Imaging of Drosophila Larval Neuroblasts

的实时成像<em&gt;果蝇</em&gt;幼虫神经母细胞

该协议详细说明了用于进行活细胞成像在一个完整的幼体脑的情况下简化方法。活细胞成像的方法是无价的，不对称的神经干细胞分裂的研究以及其他神经和发育过程，坚持揭露先前忽略的机制。

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Research

JoVE Journal

Neuroscience

Live Imaging of Drosophila Larval Neuroblasts

Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning

Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning

生活磁共振波谱果蝇</em用魔角旋转

High-Resolution Magic Angle Spinning (HRMAS) proton magnetic resonance spectroscopy (1H-MRS) is a novel non-destructive technique that improves spectral ...

科研

神经科学

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

3幼虫节段性神经的可视化 RD龄果蝇幼虫的筹备工作

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

在果蝇幼虫分部轴突在体内的突触小泡的可视化

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

幼虫，蛹和成人的解剖和免疫组化<em&gt;果蝇</em&gt;视网膜

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

Functional Analysis of the Larval Feeding Circuit in Drosophila

Functional Analysis of the Larval Feeding Circuit in Drosophila

在幼虫馈电电路的功能分析果蝇</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

为什么量化事项：表型的表征在<em&gt;果蝇</em&gt;幼虫神经肌肉接头

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

用线性琼脂糖渠道研究<em&gt;果蝇</em&gt;幼虫爬行行为

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

单细胞分辨荧光活成像在幼虫脑培养中的果蝇昼夜节律时钟

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

跟踪果蝇幼虫行为对嗅神经元 Optogenetic 刺激的反应

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...