我们感谢博士。裕农扬和韦斯Grueber提供援助，带LCM飞的股票，在本研究中使用的，弗吉尼亚Espina，伊曼纽尔Petricoin博士和博士兰斯利奥塔。作者承认这项研究的支持（DNC）和乔治梅森大学教务长办公室（EPRI）的托马斯F和凯特米勒Jeffress纪念信托。

对果蝇 LCM的外周神经元的一般意见允许来自6个小时，一个星期或液晶显示模块不再取决于组织类型和所需的细胞数量。 所有的程序都进行严格的无RNase条件以下的标准程序。幼虫表达无论是21 - 7 - GAL4，无人机mCD8：：GFP或PPK - GAL4 UAS - mCD8：：GFP转基因记者线，这些实验中使用。 1。准备幼虫<ol><li>收集30-40年龄相匹配的第三龄幼虫和DDH 2 O冲洗，随后进行了简短的在客场核糖核酸酶（Sigma - Aldrich公司）冲洗，最后洗删除核糖核酸酶在DDH 2 。威克关闭多余的DDH 2 O完全用干净的Kimwipe事先嵌入在华侨城的幼虫。 </li><li>以干净的纸巾嵌入模具和一层薄（1.5毫米），华侨城层，就足以覆盖单层的幼虫。 </li><li>嵌入到幼虫之前，如果需要的话，冷却至0℃，在组织包埋模具的OCT。请放在模具上的冰块含有华侨城。这将有助于减少在嵌入过程中的幼虫的运动。 </li><li>将预冷OCT干净的幼虫，并安排他们在彼此平行。一旦幼虫已安排，慢慢填补，而不会干扰幼虫的安排与华侨城的模具。对齐冻结放在干冰块模具。 [ 关键的一步 ：扣冻结的幼虫立即减少运动]，此方法将允许幼虫在一个平面上，最大限度地发挥每节为捕捉细胞数量。 如果有必要保留识别感兴趣的细胞组织形态，或预计每节细胞数，如果发现是令人满意的，然后直接进行步骤11。 另外，如果细胞是专门标记，并可以用一个易于识别的标记，如GFP或RFP确定，但每节细胞的数量是低，那么步骤5-10，可能有助于增加可用的细胞数量捕捉每节。这些都是可选的步骤，并应考虑作为增加DA能神经元的数目，每节的LCM的方法，其他方法失败时，提供了有利的结果，只有当所需。 [ 注意 ：此方法可能会破坏整体的组织形态和组织确定具体的空间位置上的非常困难。] </li><li> 50-70第三龄幼虫在第1步所述洗净。将在1.5 ml离心管中含有500μl的RNase免费1X PBS的幼虫。 </li><li> Dounce使用6-7缓慢强大的招聚丙烯杵（美国科学），幼虫。 </li><li>离心16,000解决方案（X）5-10秒G（安顿下来，直到幼虫表皮离心管底部。弃上清。沉淀应主要包括幼虫角质层的期票，包括DA能神经元，严格遵守。 </li><li>在1X PBS重新悬浮颗粒在500μL1 × PBS清洗角质层颗粒的2-3倍。自旋16,000（X）1分钟，形成一个紧凑颗粒克的解决方案。完全用细巴斯德吸管吸出上清液。 </li><li>小心取出离心管使用一个干净的铲的颗粒，灯芯关闭多余的PBS使用一个干净的Kimwipe组织。 [ 注 ：重要的是要尽可能的紧凑颗粒提高细胞产量]。紧凑角质层颗粒放在一个塑料模具含有一层薄薄的华侨城（1毫米约）。 </li><li>轻轻地摊开沉淀在薄薄的圆形区域。与华侨城填充模具，冻结的组织，如步骤4所述。 </li><li>使用低温恒温器，切割冻结在5-8微米厚的部分平原，标记，不带电，核糖核酸酶的玻璃显微镜玻片上。 [ 关键的一步 ：幻灯片的中心位置，靠近组织切片。] </li><li>商店可以直接在-80 ° C在一个干净的滑框，直到准备显微切割为干冰或幻灯片。执行LCM的一个星期内，最好在切片组织。 </li></ol> 暂停点 2。脱水和角质层去除冷冻幼体第 [ 关键的一步 ：脱水所有解决方案都必须准备在每个LCM会议新鲜。实现最佳显微切割效率是必不可少的幼虫冰冻组织切片的完全脱水] <ol><li>删除包含从-80 ° C冰箱的冷冻幼体组织切片的幻灯片，并放置在干冰。 </li><li>删除单个幻灯片和干冰立即直接放入50毫升锥形TU填写由核糖核酸酶自由DDH 2 O冲洗，根据表1中所建议的时间很短，用70％乙醇固定液。 </li><li>在冰冻切片幼虫角质层的存在，可能会损害防止高效的LCM第细胞接触，这可能会导致非特异性捕获的捕获效率。如有必要，进行下列步骤（4-5），从组织切片以清除角质层。 </li><li>轻轻移液器50μL2.5％胰蛋白酶直接到组织切片，并在室温下孵育5-30秒。 [ 关键的一步 ：这一步可能需要优化。培养时间取决于要显微切割的组织和切片厚度。潜伏期较长，可能会清除一切，包括细胞的利益，而一个潜伏期短，可能不足以去除角质层] </li><li>各节简要冲洗在50 ml锥形管含无RNase DDH 2去除胰蛋白酶，和松散坚持角质层碎片。 </li><li>浸在剩余的乙醇和二甲苯所建议的时间（见表1）解决方案，每年的幻灯片顺序完成脱水过程。 </li><li>以下梯度脱水，幻灯片简要干为60-120秒执行的LCM前在室温下温和的空气流。 [ 注意 ：干二甲苯完全从组织切片前执行的LCM。二甲苯被称为解散LCM第聚合物在显微切割技术故障造成的]。 </li></ol><table border="1"><tbody><tr><td> 70％乙醇（固定液） </td><td> 3-10秒</td></tr><tr><td> DDH 2 Ø </td><td> 5-10秒</td></tr><tr><td> 2.5％胰蛋白酶孵化 </td><td> 5-30秒</td></tr><tr><td> DDH 2 Ø </td><td> 5-10秒</td></tr><tr><td> 70％的乙醇 </td><td> 60秒</td></tr><tr><td> 95％的乙醇 </td><td> 60秒</td></tr><tr><td> 100％的乙醇 </td><td> 120秒</td></tr><tr><td> 100％的乙醇 </td><td> 120秒</td></tr><tr><td> 100％二甲苯 </td><td> 120秒</td></tr><tr><td> 100％二甲苯 </td><td> 120秒</td></tr><tr><td> 空气干燥温和的空气流 </td><td> 60-120秒</td></tr></tbody></table> 表1：推荐幼虫冰冻组织切片的LCM脱水过程 。 3。激光捕获显微切割 PixCell IIe上LCM与EGFP的优化Fluor公司300 epifluorescence光学配备的仪器是用于表演的LCM。 [ 关键的一步 ：如果可能的话，执行的显微切割技术，湿度控制的房间，以防止任何显微切割效率，减少因湿度增加。房间湿度是影响显微切割效率的一个关键因素。室内湿度低，可能导致增加的静电附着导致非特异性捕获，而室内湿度高，可能会导致在显微切割效率低。我们取得了25-50％之间，相对湿度条件下的最佳LCM的效率。] <ol><li>显微镜和激光控制箱接通电源。 </li><li> HS LCM的上限（Molecular Devices公司）与负载CapSure第持有人大会：LCM的帽盒可能被加载一次。 </li><li>打开分子器件和软件，进入实验的细节，包括幻灯片编号和第批号。 </li><li>到PixCell IIe上LCM仪表和操纵杆的位置是正确的墨盒装入一个新的HS LCM的第有关上限，以确保正确的定位，以捕获区域。 </li><li>置于显微镜幻灯片，其中包含新鲜脱水幼体组织切片的显微解剖显微镜阶段。 </li><li>找到使用的目镜或计算机屏幕上的荧光标记的细胞。由于高特异性PPK - GAL4，UAS - mCD8：GFP转基因记者线，IV级DA能神经元可以很容易地确定了GFP荧光。 </li><li>受组织LCM使用CapSure协LCM帽一套详细的指令用于设置仪器，聚焦的激光和表演的LCM见参考文献22]。 </li><li>调整“电源”和“持续时间”激光脉冲参数，以实现精确的聚合物熔体的斑点，其大小对应于选定的激光光斑大小。调整设置自定义的聚合物熔体面积的大小。以下参数用于microdissecting单IV级DA能神经元：光斑直径7.5微米，激光强度为30-50毫瓦，激光时间和2-4 MS和细胞每1-2张。 [ 关键的一步 ：一个适当的地点所需的激光参数可能会有所不同组织的厚度和房间的湿度条件。调整的“权力”和“持续时间”设置来实现为microdissecting单个或多个细胞所需的聚合物熔体光斑大小。] </li><li>为了最大限度地提高特异性，以最小的重叠和较强的荧光microdissect细胞。每个CAP是能够捕捉大量的细胞体。 [ 关键的一步 ：为了避免RNA降解，限制总的分析时间不少于45分钟，包括脱水和显微切割] </li><li>一旦所有感兴趣的细胞已被抓获，解除与显微切割细胞的HS LCM的上限，并放置在一个干净的幻灯片，以确认捕获细胞的存在，并目视检查不需要的细胞或碎片的第。 </li></ol> 4。从LCM源性细胞的RNA隔离<ol><li>包含显微切割细胞连接的第一个ExtracSure（Molecular Devices公司）设备。添加到第表面含有细胞的RNA提取缓冲液12μl（PicoPure，分子器件）和一个倒置的薄壁反应管连接（GeneAmp，美国应用生物系统公司）的ExtracSure设备。将对齐托盘（Molecular Devices公司）的大会，并与预先加热至42 ° C内的孵化器孵化块（Molecular Devices公司）覆盖。 </li><li>经过30分钟的孵育，离心管包含在800（X）G提取物为2分钟。关闭管和细胞提取物储存于-80 °，直到准备RNA纯化彗星。 </li></ol> 暂停点 <ol start="3"><li>提取和柱纯化的RNA按照PicoPure（Molecular Devices公司）RNA提取试剂盒说明书。 DNA酶处理是可选的，可以列上执行，在必要的RNA纯化。如果需要，可以汇集多个LCM的样品在单个列的净化，增加最终的RNA产量，并且可以在一个小体积的洗脱缓冲液（11-30μL）（PicoPure，分子器件）洗脱，并保存于-80 ° C，直到准备使用。如果需要一个1μL等份可用于评估一个2100生物分析仪（安捷伦科技公司）的总RNA质量。 </li></ol>代表性的成果激光捕获显微切割（LCM）的用于隔离单类特定或多个的果蝇DA能神经元，从第三龄幼虫（图1 ）。 LCM液晶显示模块允许在果蝇的大神经元胞体（图2A - C）的隔离程度高的精确度和特异性。此外，从这些孤立的DA能神经元纯化总RNA被发现的优秀品质急剧5.8S，18S和28S核糖体RNA峰的存在表明，安捷伦2100生物分析仪（安捷伦科技公司）（图分析2D）。 为了评估特定的浓缩，分离的细胞神经元，我们进行实时定量逆转录聚合酶链反应（QRT - PCR）使用的神经元基因的特定标记，elav。对于定量RT - PCR分析，我们计算显微切割的DA能神经元和规范化的这种表达我们的内对照（rp49）从整个幼虫使用ΔΔCT 方法 23分离的总RNA相对的LCM elav表达的相对水平。这些分析显示，在LCM的elav的相对水平的2.5倍浓缩微解剖DA神经元的样品相比，整体动物（图3 ）。 <img src="/files/ftp_upload/2016/2016fig1.jpg" alt="图1" /> 图1： 果蝇外周神经元的液晶显示模块 。 （一）年龄匹配的第三龄幼虫的选择，洗涤及（b）在华侨城cryomold嵌入式和冷冻在-80 ° C（C）8微米的串行组织切片使用一个标准的低温恒温器和（d）放置均匀清洁玻片。贴的组织切片与玻片储存于-80 ° C之前，液晶显示模块处理（E，F）紧接前面的液晶显示模块，组织切片解冻，简单地固定在70％的乙醇进行了简短的DDH 2 O冲洗（G）的幻灯片简要培养用胰蛋白酶和DDH 2 O冲洗去除角质层（黑）从幼虫部分（H，I）没有幼虫角质层组织切片，然后在乙醇梯度脱水，最后清除在二甲苯（j）条 LCM与耐热聚合物帽放在组织切片和激光脉冲是在选定的荧光细胞体。激光脉冲熔化的聚合物和吞蚀选定的细胞体。聚合物帽一起捕获的细胞体，解除， （K）RNA的提取液添加到单元格。捕获的细胞与RNA提取缓冲液，42℃孵育30分钟，可以是储存在-80 ° C，或直接用于RNA纯化处理。 <img src="/files/ftp_upload/2016/2016fig2.jpg" alt="图2" /> 图2：LCM的促进DA能神经元的高精度捕获 。 （一）代表的脱水和胰蛋白酶的图像处理执行的LCM前8微米的组织切片，显示两个类IV DA能神经元与GFP标记的PPK - GAL4，UASmCD8：GFP报告应变。 （三）最终的注意，突出显示的神经元（箭头）捕捉（二）突出显示的神经元细胞（箭头）的身体是干净的微解剖与高特异性的组织切片。查看一个类- IV DA神经元捕获LCM的第（四）安捷伦2100生物分析仪（安捷伦科技公司）电泳派生的LCM - DA能神经元分离的总RNA，呈现出良好的RNA的质量存在尖锐的5.8S，18S ，而28S rRNA的峰。 <img src="/files/ftp_upload/2016/2016fig3.jpg" alt="图3" /> 图3：QRT - PCR揭示大量富集抓获相对于整个动物的DA能神经元的神经标记基因在LCM的表达，神经元特异性标记基因的表达在LCM（elav）QRT - PCR分析捕获的DA 能神经元（21 - 7 - GAL4， UAS - mCD8：：GFP）的整个动物的年龄相匹配的第三龄幼虫进行一式三份。 elav在LCM的捕获DA能神经元表达的相对水平正常化内对照（rp49），相对于整个幼虫使用ΔΔCT 方法 23 。一个elav在LCM的相对水平的2.5倍浓缩捕捉观察到DA神经元的样品相对于整个动物。 故障排除问题：RNA的降解，低质量或。 确保在整个实验的核糖核酸酶自由状态。尝试减少金额每张幻灯片LCM上花30分钟的时间。除了在表演LCM时保持干冰的幻灯片和样本。避免解冻组织切片，一旦他们被切断。 问题：大多数细胞是从幻灯片中失去胰蛋白酶处理（步骤17-18）。 尝试减少胰蛋白酶处理的时间。尝试减少胰蛋白酶的浓度。 问题：角质层和其他非特定的组织碎片上的第聚合物的存在。 尝试删除印迹聚合物表面与粘合剂附注22的粘性组织碎片。 问题：LCM的效率低。 尝试增加100％的乙醇和二甲苯的潜伏期时间。使用除湿机降低室内湿度。

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mRNA+from+specific+tissues+of+Drosophila+by+mRNA+tagging.">Isolation of mRNA from specific tissues of Drosophila by mRNA tagging.</a> Nucl. Acids Res. 33, (2005).</li><li>Appay, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+Gene+Expression+Profiling+of+Human+T+Cell+Subsets+Reveals+Parallel+Post-Thymic+Differentiation+for+CD4++and+CD8++Lineages.">Sensitive Gene Expression Profiling of Human T Cell Subsets Reveals Parallel Post-Thymic Differentiation for CD4+ and CD8+ Lineages.</a> J. Immunol. 179, 7406-7414 (2007).</li><li>Ginzinger, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+quantification+using+real-time+quantitative+PCR:+An+emerging+technology+hits+the+mainstream.">Gene quantification using real-time quantitative PCR: An emerging technology hits the mainstream.</a> Exp. Hematol. 30, 503-512 (2002).</li><li>Keays, K. M., Owens, G. P., Ritchie, A. M., Gilden, D. H., Burgoon, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+and+single-cell+RT-PCR+without+RNA+purification.">Laser capture microdissection and single-cell RT-PCR without RNA purification.</a> J. Immunol. Methods. 302, 90-98 (2005).</li><li>Volgin, D. V., Swan, J., Kubin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+RT-PCR+gene+expression+profiling+of+acutely+dissociated+and+immunocytochemically+identified+central+neurons.">Single-cell RT-PCR gene expression profiling of acutely dissociated and immunocytochemically identified central neurons.</a> J. Neurosci. Methods. 136, 229-236 (2004).</li><li>Emmert-Buck, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection.">Laser capture microdissection.</a> Science. 274, 998-1001 (1996).</li><li>Xiao, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiling+in+embryonic+mouse+lenses.">Gene expression profiling in embryonic mouse lenses.</a> Mol. Vis. 12, 1692-1698 (2006).</li><li>Scharschmidt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+human+osteoarthritic+connective+tissue+by+laser+capture+microdissection+and+QRT-PCR.">Analysis of human osteoarthritic connective tissue by laser capture microdissection and QRT-PCR.</a> Connect. Tissue Res. 48, 316-323 (2007).</li><li>Spletter, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=regulates+Drosophila+olfactory+projection+neuron+identity+and+targeting+specificity.">regulates Drosophila olfactory projection neuron identity and targeting specificity.</a> Neural Dev. 2, 14-14 (2007).</li><li>Hoopfer, E. D., Penton, A., Watts, R. J., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+analysis+of+Drosophila+neuronal+remodeling:+a+role+for+the+RNA-binding+protein+Boule+as+a+negative+regulator+of+axon+pruning.">Genomic analysis of Drosophila neuronal remodeling: a role for the RNA-binding protein Boule as a negative regulator of axon pruning.</a> J. Neurosci. 28, 6092-6103 (2008).</li><li>Lee, T., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+analysis+with+a+repressible+cell+marker+for+studies+of+gene+function+in+neuronal+morphogenesis.">Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis.</a> Neuron. 22, 451-461 (1999).</li><li>Grueber, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projections+of+Drosophila+multidendritic+neurons+in+the+central+nervous+system:+links+with+peripheral+dendrite+morphology.">Projections of Drosophila multidendritic neurons in the central nervous system: links with peripheral dendrite morphology.</a> Development. 134, 55-64 (2007).</li><li>Song, W., Onishi, M., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+multidendritic+sensory+neurons+are+necessary+for+rhythmic+locomotion+behavior+in+Drosophila+larvae.">Peripheral multidendritic sensory neurons are necessary for rhythmic locomotion behavior in Drosophila larvae.</a> Proc. Natl. Acad. Sci. USA. 104, 5199-5204 (2007).</li><li>Espina, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection.">Laser capture microdissection.</a> Nat. Prot. 1, 586-603 (2006).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method.</a> Methods. 25, 402-408 (2001).</li></ol>

本协议描述了我们对果蝇通过LCM的外周神经元的隔离的优化方法。虽然这LCM协议旨在为特定的隔离单，类特定或多个果 ​​蝇的大神经元从3龄幼虫阶段的发展，稍作修改的协议可以很容易地适用于其他果蝇的细胞类型的捕捉从所有发展使用不同的GAL4的阶段 ， 记者UAS - mCD8 - GFP转基因标签的细胞类型或类型的利益。我们的研究结果表明，激光捕获微解剖DA?...

设备： <ul><li>低温恒温器</li><li> 50 ml锥形管幻灯片固定，漂洗，胰蛋白酶处理和乙醇/二甲苯脱水</li><li> -80 ° C冰箱</li><li>孵化器</li><li> PixCell IIe上LCM仪器与福陆公司的300 epifluorescence光学优化EGFP（分子器件，分子器件） </li></ul>

laser capture microdissection of drosophila peripheral neurons

树突状树枝状（DA）神经元的果蝇周围神经系统（PNS）在调查的基础类特定的树突形态发生 1,2的分子机制提供一个极好的的模型系统。为了方便类特定的DA神经元发展的分子生物学分析，至关重要的是在一个纯粹的人口获得这些细胞。虽然一系列不同的细胞和组织特异性的RNA分离技术中存在的果蝇细胞，包括基于磁珠细胞净化3,4，荧光激活细胞分选（FACS）5-8，和RNA结合蛋白为基础的战略9 ，概无这些方法可以很容易地利用空间精度与高度隔离的单个或多个类特定的果蝇DA能神经元。激光捕获显微切割（LCM）已成为一个非常强大的工具，可以用来从组织切片中分离出特定的细胞类型，具有高度的空间分辨率和精度。然后，可以从分离的细胞获得的RNA用于包括在一个给定的细胞类型10-16的QRT - PCR和基因芯片表达谱分析。迄今为止，液晶显示模块没有被广泛应用在果蝇的组织和细胞17,18，包括发展第三龄幼虫阶段的DA能神经元的分析。 在这里，我们目前对果蝇的大神经元使用的红外（IR）类LCM的隔离，我们优化的协议。这种方法允许单一，类特定或多个的DA能神经元具有高特异性和空间分辨率的捕捉。年龄相匹配的三龄幼虫表达的UAS - mCD8：绿色荧光蛋白 19基因的控制下的IV级DA神经元具体PPK - GAL4 20驱动程序或泛DA神经元特异性21 - 7 - GAL4 21驱动程序是用于这些实验。从孤立的DA能神经元获得的RNA是非常高的质量，并可以直接用于下游应用，包括QRT - PCR或芯片分析。此外，这种液晶显示模块协议，可以方便地用于捕捉到其他果蝇的细胞类型的不同阶段的发展依赖于具体，GAL4驱动 GFP的表达模式的细胞类型的。

Watch this Scientific Journal Video about Laser Capture Microdissection of Drosophila Peripheral Neurons at JoVE.com

Laser Capture Microdissection of Drosophila Peripheral Neurons

在这个视频文章中，我们目前的方法隔离单个或多个<em&gt;果蝇</em&gt; DA神经元使用红外线（IR）的捕获激光捕获显微切割（LCM）的类三龄幼虫。从孤立的神经元获得的RNA可以很容易地用于下游应用，包括QRT - PCR或芯片分析。

激光捕获显微切割技术的果蝇外周神经元

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

laser-capture-microdissection-of-drosophila-peripheral-neurons

Research

JoVE Journal

Neuroscience

Laser Capture Microdissection of Drosophila Peripheral Neurons

13.4K 次观看.  George Mason University. 在这个视频文章中，我们目前的方法隔离单个或多个果蝇 DA神经元使用红外线（IR）的捕获激光捕获显微切割（LCM）的类三龄幼虫。从孤立的神经元获得的RNA可以很容易地用于下游应用，包括QRT - PCR或芯片分析。

视频: Laser Capture Microdissection of Drosophila Peripheral Neurons

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

形态分析果蝇幼虫外周感觉神经元树突和轴突使用遗传马赛克

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

科研

神经科学

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

总RNA的分离和下游下一代测序和基因表达谱的非激光捕获显微切割离散小鼠脑地区显微镜方法

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

激光捕获显微切割的丰富种群的创伤性脑损伤后神经元或单个神经元的基因表达分析

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.

Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.

神经元的分化的人类Neuroprogenitor细胞培养激光捕获显微切割

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

通过激光捕获微分路，从小鼠脊髓运动神经细胞体中生成RNA进行转录分析

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium carbonate. Animal studies as well as human genetic studies indicate that lithium affects molecular targets that are involved in neuronal growth, survival and maturation, and notably molecules involved in Wnt signaling. Given the ethical challenge to obtaining brain biopsies for investigating dynamic molecular changes associated with lithium-response in the central nervous system (CNS), one may consider the use of neurons obtained from olfactory tissues to achieve this goal.The olfactory epithelium contains olfactory receptor neurons at different stages of development and glial-like supporting cells. This provides a unique opportunity to study dynamic changes in the CNS of patients with neuropsychiatric diseases, using olfactory tissue safely obtained from nasal biopsies. To overcome the drawback posed by substantial contamination of biopsied olfactory tissue with non-neuronal cells, a novel approach to obtain enriched neuronal cell populations was developed by combining nasal biopsies with laser-capture microdissection. In this study, a system for investigating treatment-associated dynamic molecular changes in neuronal tissue was developed and validated, using a small pilot sample of BD patients recruited for the study of the molecular mechanisms of lithium treatment response.

In this study, a novel platform to investigate intraneuronal molecular signatures of treatment response in bipolar disorder (BD) was developed and validated. Olfactory epithelium from BD patients was obtained through nasal biopsies. Then laser-capture microdissection was combined with Real Time RT PCR to investigate the molecular signature of lithium response in BD.

通过鼻腔活检获得联合激光捕获显微切割的嗅觉神经元：一种可能的方法来研究治疗反应的精神障碍

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

激光捕获显微切割 - 个人多巴胺神经元的分离和整个腹侧被盖区的示范

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

立体定向激光捕获创伤性脑区显微解剖

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

激光捕获显微切割分离新鲜冷冻人体肠神经节的优化

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, microvascular proliferation, mesenchymal transformation and necrosis characterize the histological heterogeneity of high-grade gliomas. Our laboratory has demonstrated that the presence of high densities of mesenchymal cells, named oncostreams, correlate with tumor malignancy. We have developed a unique approach to understand the mechanisms that underlie glioma&#39;s growth and invasion. Here, we describe a comprehensive protocol that utilizes laser capture microdissection (LMD) and RNA sequencing to analyze differential mRNA expression of intra-tumoral heterogeneous multicellular structures (i.e., mesenchymal areas or areas of tumor invasion). This method maintains good tissue histology and RNA integrity. Perfusion, freezing, embedding, sectioning, and staining were optimized to preserve morphology and obtain high-quality laser microdissection samples. The results indicate that perfusion of glioma bearing mice using 30% sucrose provides good morphology and RNA quality. In addition, staining tumor sections with 4% Cresyl violet and 0.5% eosin results in good nuclear and cellular staining, while preserving RNA integrity. The method described is sensitive and highly reproducible and it can be utilized to study tumor morphology in various tumor models. In summary, we describe a complete method to perform LMD that preserves morphology and RNA quality for sequencing to study the molecular features of heterogeneous multicellular structures within solid tumors.

Laser microdissection (LMD) is a sensitive and highly reproducible technique that can be used to uncover pathways that mediate glioma heterogeneity and invasion. Here, we describe an optimized protocol to isolate discrete areas from glioma tissue using laser LMD followed by transcriptomic analysis.

Methods Article

激光捕获显微切割技术的果蝇外周神经元