我们感谢加里Laevsky对显微镜有益的讨论。这项工作是由美国国立卫生研究院资助的授予R01GM061107和R01GM067758到ERG

注意:对于肌肉切除（ 图1）的步骤的制备幼虫鱼片先前描述的方法的变形例。之前和之后的肌肉除去步骤概述简要地并且读者可以参考以前的工作10，11，用于更详细的描述。 1.解剖幼虫在冷盐水<ol><li>准备冷HL3.1生理盐水15或冷的Ca 2+ HL3.1生理盐水11（ 表1）的工作稀释。放置幼虫与刚好足够冷盐水硅氧烷弹性体盘，以覆盖培养皿的底部。 注:请参阅有关生理盐水的选择讨论。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 1X HL3.1盐水（pH 7.2） </td></tr><tr><td> 5毫米的HEPES </td></tr><tr><td> 70毫米氯化钠</td></tr><tr&gt; <td> 5毫米氯化钾</td></tr><tr><td> 1.5毫米氯化钙2（省略无钙生理盐水） </td></tr><tr><td> 4毫米氯化镁2 </td></tr><tr><td> 10毫米碳酸氢钠 </td></tr><tr><td> 5毫米海藻糖</td></tr><tr><td> 115毫米蔗糖</td></tr><tr><td></td></tr><tr><td>过滤消毒和储存在4℃ </td></tr><tr><td>注:作文模仿昆虫的血淋巴</td></tr></tbody></table> 表1.冷盐水的组成。 <ol start="2"><li>与腹侧幼虫位置。幼虫的腹面可以通过运行由前至后的主幼虫气管导管腹部dentical皮带和背侧被识别。伸展幼虫在前后方向和头尾针到一个有机硅弹性体用昆虫针解剖盘。用细解剖剪刀沿腹中线切开，在一端开始向另一进展。 注:此方位保留DA能神经元的常用研究背集群。 </li><li>幼虫切开后，PIN四角的解剖盘，就像打开一本书。使用镊子抓住并取出内脏器官，包括中枢神经系统，肠道，和气管。调整昆虫针，使圆角绷紧但不是最大延伸。 注:肌肉是在段的边界固定于体壁。虽然肌肉覆盖大部分体壁的，他们是在靠近背中线的狭窄的区域不存在。 </li></ol> 2.去除肌肉<ol><li>找到幼虫，其中肌肉组织缺席的背中线。位置的单个钳子尖头，使得其可插入在尽可能平坦的取向的肌肉组织的下方。 </li><li>开始于背中线，该段的前边界附近，小心地将肌肉和表皮之间的钳子叉，注意尽量减少镊子和表皮之间的接触。 </li><li>向上拉钳子打破肌肉体壁的附着在一个锚定点。重复此过程针对感兴趣的剩余半段（多个）。 注:此协议优化了每个大神经元树突场后的保存。为了保持枝晶场的前部，最好是在各段的后端插入镊子叉。 </li><li>重新调整昆虫针，使得幼虫鱼片在所有方向最大限度拉伸。 </li><li>修复圆角而这是采用冷，新鲜烹制的PBS中的4％甲醛25分钟还是被钉在解剖盘。 </li><li>在PBS中漂洗5次。 </li><li>使用镊子从剩余的锚点仔细扯远了肌肉组织，同时注意尽量减少CONTA克拉与表皮。 </li><li>取消固定，并从解剖盘取出鱼片到1.5ml微量离心管中。执行所有随后的洗涤，阻挡，和免疫荧光染色步骤，如前所述11。 </li></ol> 3.安装幼虫鱼片<ol><li>首先用细解剖剪，以使样品尽可能平坦去除头部和尾部。安装在盖玻片圆角与面对盖玻片圆角的内表面抗褪色封固剂。 </li><li>广场上的盖玻片显微镜载玻片，并轻轻按压以驱散安装介质。翻转显微镜载玻片和密封使用指甲油幻灯片盖玻片。玻片可以储存在-20℃下至少一个月。 </li></ol>

<ol>
	<li>Jan, Y. N., Jan, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Branching+out:+mechanisms+of+dendritic+arborization.">Branching out: mechanisms of dendritic arborization.</a> Nat Rev Neurosci. 11 (5), 316-328 (2010).</li><li>Corty, M. M., Matthews, B. J., 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=Molecules+and+mechanisms+of+dendrite+development+in+Drosophila.">Molecules and mechanisms of dendrite development in Drosophila.</a> Development. 136 (7), 1049-1061 (2009).</li><li>Parrish, J. Z., Emoto, K., Kim, M. D., 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=Mechanisms+that+regulate+establishment,+maintenance,+and+remodeling+of+dendritic+fields.">Mechanisms that regulate establishment, maintenance, and remodeling of dendritic fields.</a> Annu Rev Neurosci. 30, 399-423 (2007).</li><li>Kim, M. E., Shrestha, B. R., Blazeski, R., Mason, C. A., 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=Integrins+establish+dendrite-substrate+relationships+that+promote+dendritic+self-avoidance+and+patterning+in+Drosophila+sensory+neurons.">Integrins establish dendrite-substrate relationships that promote dendritic self-avoidance and patterning in Drosophila sensory neurons.</a> Neuron. 73 (1), 79-91 (2012).</li><li>Han, C., 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=Integrins+regulate+repulsion-mediated+dendritic+patterning+of+Drosophila+sensory+neurons+by+restricting+dendrites+in+a+2D+space.">Integrins regulate repulsion-mediated dendritic patterning of Drosophila sensory neurons by restricting dendrites in a 2D space.</a> Neuron. 73 (1), 64-78 (2012).</li><li>Parrish, J. Z., Xu, P., Kim, C. C., 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=The+microRNA+bantam+functions+in+epithelial+cells+to+regulate+scaling+growth+of+dendrite+arbors+in+Drosophila+sensory+neurons.">The microRNA bantam functions in epithelial cells to regulate scaling growth of dendrite arbors in Drosophila sensory neurons.</a> Neuron. 63 (6), 788-802 (2009).</li><li>Jiang, N., Soba, P., Parker, E., Kim, C. C., Parrish, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microRNA+bantam+regulates+a+developmental+transition+in+epithelial+cells+that+restricts+sensory+dendrite+growth.">The microRNA bantam regulates a developmental transition in epithelial cells that restricts sensory dendrite growth.</a> Development. 141 (13), 2657-2668 (2014).</li><li>Meltzer, 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=Epidermis-derived+Semaphorin+promotes+dendrite+self-avoidance+by+regulating+dendrite-substrate+adhesion+in+Drosophila+sensory+neurons.">Epidermis-derived Semaphorin promotes dendrite self-avoidance by regulating dendrite-substrate adhesion in Drosophila sensory neurons.</a> Neuron. 89 (4), 741-755 (2016).</li><li>Han, C., 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=Epidermal+cells+are+the+primary+phagocytes+in+the+fragmentation+and+clearance+of+degenerating+dendrites+in+Drosophila.">Epidermal cells are the primary phagocytes in the fragmentation and clearance of degenerating dendrites in Drosophila.</a> Neuron. 81 (3), 544-560 (2014).</li><li>Brent, J. R., Werner, K. M., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+larval+NMJ+dissection.">Drosophila larval NMJ dissection.</a> J Vis Exp. (24), (2009).</li><li>Karim, M. R., Moore, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+analysis+of+Drosophila+larval+peripheral+sensory+neuron+dendrites+and+axons+using+genetic+mosaics.">Morphological analysis of Drosophila larval peripheral sensory neuron dendrites and axons using genetic mosaics.</a> J Vis Exp. (57), e3111 (2011).</li><li>Gustafsson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surpassing+the+lateral+resolution+limit+by+a+factor+of+two+using+structured+illumination+microscopy.">Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy.</a> J Microscopy. 198 (2), 82-87 (2000).</li><li>Ashdown, G. W., Cope, A., Wiseman, P. W., Owen, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+flow+quantified+beyond+the+diffraction+limit+by+spatiotemporal+image+correlation+of+structured+illumination+microscopy+data.">Molecular flow quantified beyond the diffraction limit by spatiotemporal image correlation of structured illumination microscopy data.</a> Biophys J. 107 (9), L21-L23 (2014).</li><li>Zhang, W., Yan, Z., 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=Sound+response+mediated+by+the+TRP+channels+NOMPC,+NANCHUNG,+and+INACTIVE+in+chordotonal+organs+of+Drosophila+larvae.">Sound response mediated by the TRP channels NOMPC, NANCHUNG, and INACTIVE in chordotonal organs of Drosophila larvae.</a> PNAS. 10 (33), 13612-13617 (2013).</li><li>Feng, Y., Ueda, A., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+minimal+hemolymph-like+solution,+HL3.1,+for+physiological+recordings+at+the+neuromuscular+junctions+of+normal+and+mutant+Drosophila+larvae.">A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae.</a> J Neurogenet. 18 (2), 377-402 (2004).</li><li>Babcock, D. T., Landry, C., Galko, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+signaling+mediates+UV-induced+nociceptive+sensitization+in+Drosophila+larvae.">Cytokine signaling mediates UV-induced nociceptive sensitization in Drosophila larvae.</a> Curr Biol. 19 (10), 799-806 (2009).</li></ol>

作者什么都没有透露。

在这里，一个协议是从的果蝇幼虫鱼片手工清除肌肉组织的描述。该协议会修改先前描述的幼虫解剖技术10,11。幼虫是在有机硅弹性体解剖的菜后，背中线的位置。一个单一的镊子尖头在其尽可能平坦的方向，仔细插入肌肉组织和表皮之间，靠近背中线。镊子轻轻向上从一个锚点中的每个感兴趣的幼虫段被拉至单独的肌肉组织。然后幼虫鱼片被固定在冷甲醛之后钳子再次用于从剩余的锚点抽离肌肉。 虽然这是很有诱惑力的执行肌肉拆卸步骤后固定的全部，我们发现，拉伸肌肉，一旦固定，保持扁平，甚至从锚点脱离后紧密结合在表皮，呈现它很难操作而不损伤下面的组织。此外，根据我们的经验，固定的肌肉组织脆，易流泪，防止体壁其彻底清除。相反，当肌肉被从固定前1锚点分离，这将在定影期间收缩朝向其余锚点，留下可通过镊子更容易抓住并从幼虫体壁拉动组织存根。 以最好地保存样品，相当必须小心，以尽量减少肌肉切除过程期间在镊子和表皮之间的接触。其结果是，我们的协议可能在几分钟到先前描述的解剖技术10,11添加。以避免组织退化，被前固定解剖幼虫的数目应限制，使得经过时间不超过25分钟。此外，我们建议测试钳多对 - 同莫钳德尔可能在形状和清晰度相当大的变化 - 和调整的程度鱼片肌肉中取出之前，以改善组织保存和解剖速度拉伸。 最后，我们建议您用幼虫解剖过程中使用的Ca 2+ HL3.1生理盐水与标准HL3.1盐水试验。在不存在的钙，幼虫肌肉收缩都大大降低。因此，幼虫鱼片可能会更容易在无钙 HL3.1生理盐水解剖时操控。然而，我们发现，肌肉收缩的肌肉和表皮之间创造空间，这可以促进钳子的这些组织之间的插入，同时尽量减少到表皮损坏的风险。其结果，标准HL3.1可以优选用于保存表皮和多巴胺神经元的完整性。我们取得了良好的结果与盐水和选择留给用户。 虽然我们的协议增加的先前描述解剖技术10,11的持续时间和复杂性，它提供了几种改进，是用于成像幼虫的感觉神经元和表皮细胞是有用的。第一，它允许被另有肌肉组织遮蔽neuronally和epidermally表达的蛋白的成像。第二，它提高了荧光信噪比。最后，它能够在DA能神经元和表皮蛋白质空间关系的卓越歧视利用超分辨率显微镜。我们所观察到以下肌肉除去改善成像质量很可能在样品和盖玻片之间减少光子吸收和散射，以及减少后的距离的结果。虽然在这里没有证明，肌肉切除可能还提高抗体进入表皮和多巴胺神经元，提高免疫的质量。更大的抗体可访问可以是用于在艾比森进行免疫荧光协议特别有用组织渗透剂4 CE。 通过使用此协议获得的改进，可能有益于调节在感觉神经元树突形态发生神经和表皮因素的研究。此外，肌去除先前已促进了感觉神经元的电生理研究在幼虫体壁和这个协议的修改版本可以对于采用幼虫感觉神经元的神经生理学操纵未来的研究是有用的。最后，这个协议的修改版本可以是对其他应用中有用，例如单多巴胺神经元的基因表达图谱的分离。

果蝇幼虫树突分支（DA）神经元由于其顺从遗传操纵和与它们可以被成像的容易程度为研究神经元发育一个有价值的模型。这些感觉神经元已经在控制树枝状结晶形态1-3众多途径的鉴定工具。 四类DA能神经元（类别I - IV）支配幼虫表皮。这些神经元位于基底膜和表皮之间，与他们的树突形成基本上二维阵列4,5。四个班，IV级DA能神经元具有最高度支乔木，像其他动物的感觉神经元，这些乔木的制定需要从邻近组织的内在因素，以及线索，尤其是表皮，为他们的发展6-9 。 研究确定怎么这样神经元和额外的神经元的事实ORS通过免疫荧光检测原位蛋白表达的能力控制树突形态效益。幼虫的外表皮是穿不透的抗体，但这个障碍是很容易通过行之有效的夹层方法-10,11-制备幼虫鱼片克服。然而，正好位于内部到基底膜体壁肌肉组织呈现朝大神经元和表皮细胞可视若干挑战。首先，肌肉组织，这行最体壁，大大掩盖从神经元或表皮组织发出的荧光信号。这大大降低了信号与样品中的信噪比。第二，许多相关的蛋白质可以在肌肉组织中，以及在神经元或表皮来表示。此肌肉衍生的荧光信号可能从神经元或表皮荧光信号的进一步晦涩检测。最后，先进的显微技术不在亚衍射分辨率样品W的许可证成像并可能挑剔了在神经元中表达的蛋白质的定位和周围的表皮细胞12,13尤其有用。然而，通过从强信噪比和样品到盖玻片接近超高分辨率显微镜好处成像。除了降低信噪比，幼虫体壁肌肉的距离从盖玻片，从而限制可与超分辨率显微镜方法来实现改进的图像分辨率达的神经元。除了为免疫荧光分析挑战，肌肉组织提出从感觉神经元在幼虫体壁的障碍，电生理记录。因此，它有利于去除感觉神经元14的神经生理学操纵。 此处被描述为手动去除果蝇幼虫的肌肉组织的方法。我们证明了我们的协议p蛋白质ermits免疫荧光成像被另有肌肉组织遮蔽，提高了信号到第IV类多巴胺神经元的可视噪声比，并允许使用超分辨率显微镜，以更好地辨别在多巴胺神经元的蛋白质和细胞结构的空间关系和表皮。

<table><tbody><tr><td>Dumont #5 tweezers</td><td>Electron Microscopy Sciences</td><td>72701-D</td><td></td></tr><tr><td>Micro Scissors, 8 cm, straight, 5 mm blades, 0.1 mm tips</td><td>World Precision Instruments</td><td>14003</td><td></td></tr><tr><td>Sylgard 184 silicone elastomer kit</td><td>Dow Corning</td><td>3097358-1004</td><td>for dissecting plates</td></tr><tr><td>Austerlitz insect pins, 0.1 mm</td><td>Fine Science Tools</td><td>26002-10</td><td></td></tr><tr><td>Fostec 8375 light source</td><td>Artisan Technology Group</td><td>62792-4</td><td></td></tr><tr><td>Zeiss Stemi 2000</td><td>Carl Zeiss Microscopy</td><td></td><td></td></tr><tr><td>Vectashield antifade mounting medium</td><td>Vector Laboratories</td><td>H-1000</td><td>for confocal microscopy</td></tr><tr><td>Prolong Diamond antifade mountant</td><td>Life Technologies</td><td>P36970</td><td>for structured illumination microscopy</td></tr><tr><td>Micro cover glass, 22 x 22 mm, No. 1.5</td><td>VWR</td><td>48366-227</td><td></td></tr><tr><td>Superfrost Plus microscope slides, 25 x 75 x 1.0 mm</td><td>Fisherbrand</td><td>12-550-15</td><td></td></tr><tr><td>Mouse anti-Coracle antibody</td><td>Developmental Studies Hybridoma Bank</td><td>C615.16</td><td>supernatant, dilute 1:50</td></tr><tr><td>Mouse anti-Discs large antibody</td><td>Developmental Studies Hybridoma Bank</td><td>4F3</td><td>supernatant, dilute 1:50</td></tr><tr><td>Rabbit anti-dsRed antibody</td><td>Clontech</td><td>632496</td><td>dilute 1:1,000</td></tr><tr><td>Goat anti-rabbit antibody, Alexa Fluor 568 conjugated</td><td>ThermoFisher Scientific</td><td>A-11011</td><td>dilute 1:1,000</td></tr><tr><td>Goat anti-mouse antibody, Alexa Fluor 488 conjugated</td><td>ThermoFisher Scientific</td><td>A-11001</td><td>dilute 1:500</td></tr></tbody></table>

removal of drosophila muscle tissue from larval fillets for immunofluorescence analysis of sensory neurons and epidermal cells

的果蝇幼虫树突分支（DA）神经元是研究神经元形态发生机制流行模式。 DA能神经元发展与表皮细胞它们所支配，因而从免疫荧光两种neuronally和epidermally表达蛋白的原位可视化他们的分析好处沟通。制备用于免疫荧光实验幼虫鱼片的传统方法留下完好的肌肉组织，覆盖大部分的体壁，呈现几个挑战于成像神经元和表皮蛋白质。在这里，我们描述了从的果蝇幼虫鱼片去除肌肉组织的方法。该协议可允许否则由肌肉组织遮蔽蛋白成像，改进了信噪比，并方便了使用超分辨率显微镜来研究哒神经发育。

我们证明肌肉切除过程的效用改善信噪比在免疫荧光实验共同形象化隔连接蛋白小艇（科拉）和光盘大型（DLG）用标记与膜标记IV级多巴胺神经元一起CD4- tdTomato。 科拉先前已用于识别阿凡达的神经元树突是由表皮细胞封闭，是已在研究协会DA神经元的形态和功能的树突4,6-9,16许多鉴定表皮因素之一大片。用抗DsRed的免疫染色，以检测神经元（红色）和抗科拉抗体（绿色）中的与肌肉除去（肌肉断）幼虫鱼片进行与肌肉完好（肌肉上）。对于每个条件，IV级大神经元的后树突字段使用激光扫描共聚焦显微镜成像。使用ID获得的图像entical成像参数的两个条件。我们另外成像肌肉上使用增加激光功率，以补偿从肌肉组织干扰圆角。 在肌断样品，科拉可以清楚地在表皮细胞边界检测，沿IV级DA神经元树突间歇大片，并概述了IV级DA神经元胞体（ 图2C）。与此相反，其定位于这些结构域在很大程度上遮蔽在肌肉上的样品，即使在激光功率增大（ 图2A-2B）。在肌肉上的样品，科拉主要是可视化在肌肉，气管，并在神经肌肉接头（NMJ），其成为从本体壁分离出作为肌肉切除过程的结果。从这些组织发出的荧光信号遮蔽大部分定位于表皮细胞边界和在体内沃尔的窄条到枝晶束，除了科拉的L近背中线，其中肌肉不存在。科拉，概述了IV级DA神经元胞体在肌肉样本（ 图2A-2B）没有显现。 epidermally表达的蛋白质也具有很高的肌肉表现，如DLG的可视化，特别是有问题的。在肌肉样本，DLG的在表皮的分布几乎完全由荧光从肌肉和神经肌肉接头（ 图2D）遮蔽。肌肉切除后，DLG容易在表皮细胞边界检测，沿大神经元树突间歇大片，并概述了IV级DA神经元胞体（ 图2E）。因此，肌肉能去除肌肉和相关组织，否则遮挡epidermally和neuronally表达蛋白的可视化。 此外，有人指出，在第四类哒神经元标记物的荧光强度中出现减少肌肉上鱼片相比，肌肉断鱼片时成像参数保持两个样品制剂（ 图2A，2C）之间恒定。增加激光功率时，IV级大神经元标记物的表观荧光强度可以匹配到肌肉断样品但背景噪声增加（ 图2B，2C）。因此，肌肉除去改善了信号到我们的样品中的信噪比。 最后，我们测试是否通过去除肌肉组织中获得的改进可以在子衍射分辨率应用到IV类多巴胺神经元的成像。要做到这一点，三维结构照明显微镜进行（SIM）成像，达到约120纳米的横向分辨率13。至于共焦成像，我们比较免疫染色科拉和IV级DA能神经元，第一次在相同的成像条件，然后优化激光功率后，肌肉的肌断片，博览会URE时间，和图像处理对肌肉上的样品。同样地共焦显微镜相比，肌肉断鱼片（ 图3A-3C），观察到基本上降低的信号，以在肌肉上的信噪比。此外，图像重建出现肌断样品中更清晰。相比肌肉样本，观察到较少的表观工件和科拉树突大片被更容易地得到解决（ 图3B，3C）。树突膜能在宽约114纳米肌断鱼片被测量，但似乎是宽272纳米的肌肉上鱼片（ 图3D，3E）。因此，我们的协议使超分辨率显微镜到IV级DA能神经元的可视化应用。 <img alt="图1" src="/files/ftp_upload/54670/54670fig1.jpg" /> 图1.概述肌肉去除程序，幼虫被解剖并固定到硅弹性体解剖盘。要删除的肌肉组织，一是钳叉插入肌肉和表皮细胞层之间，由段边界（2.2）附近背中线开始。钳子向上拉破肌肉和体壁（2.3）之间的连接。这重复的兴趣分段，然后将幼虫被固定在甲醛（2.5）。固定后，钳子用于剩余肌肉组织从主体壁（2.7 - 2.8）分开的幼虫角质层和表皮在橙色所示，在绿色IV级大神经元和肌肉为红色。在（2.2）和（2.7）的插图表示单个幼虫半段的横截面图。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54670/54670fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54670/54670fig2.jpg" /> 图2.去除肌肉提高多巴胺神经元和科拉的表皮免疫荧光共焦成像。（绿色，AC）或DLG（绿色; DE）与肌肉组织（A，B，D），并没有肌肉组织（C，E）进行检测。 IV级大神经元使用GAL4&#39;477驱动来表达UAS-CD4标记:tdTomato并用抗DsRed的抗体（红色）检测。标记为科拉肌肉上和肌肉断样品先用相同的条件（A，C）根据共焦显微镜成像，然后增加激光功率，以补偿肌肉干扰（B）中。标示DLG肌肉上和肌肉断开样品单独优化的条件下成像。所有图像都共聚焦Z-预测。中间（绿色）和底部（红色）面板显示单个频道;顶部面板是合并的信道。肌肉组织的（虚线）的边界中，IV类DA神经元胞体（青色箭头），科拉和DLG枝晶大片（白色箭头），表皮细胞界限（品红色箭头），NMJ（黄色箭头）和气管（橙色箭头）表示。比例尺= 20微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54670/54670fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54670/54670fig3.jpg" /> 图3.肌肉后，使超分辨率DA能神经元和表皮细胞，与肌完好（A，B）和后肌肉清除（C）IV级DA能神经元免疫荧光检测科拉（绿色） 的成像 。 IV级大神经元标记为在图2中的肌肉上和肌肉断样品成像与第一下相同条件（A，C）一种结构照明显微镜。在肌肉样品在（A）中，然后用增大激光功率和曝光时间重新映像，以补偿肌肉干扰（B）中。所有图片均为Z-预测。交流的中间（绿色）和底部（红色）面板显示单独的信道。交流的顶板是一个合并。表皮细胞边界（品红箭头），科拉树枝状大片（白色箭头），以及图像重建伪影（黄色箭头）表示。在B和C的底部面板插图分别对应于D和E。枝晶膜的表观宽度示于肌肉上（D）和肌断（E）的样品。比例尺= 3微米（AC）和0.5微米（D，E）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54670/54670fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells at JoVE.com

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

使用的果蝇幼虫树突分支（DA）神经元的神经元形态的研究通过免疫荧光在神经细胞和表皮蛋白质的原位可视化中受益。我们描述通过从幼虫体壁除去肌肉组织提高多巴胺神经元和包围的表皮细胞的免疫荧光分析的过程。

去除<em&gt;果蝇</em从幼虫鱼片的感官神经元和表皮细胞的免疫荧光分析&gt;肌肉组织

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

removal-drosophila-muscle-tissue-from-larval-fillets-for

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

8.9K 次观看.  Princeton University. 使用的果蝇幼虫树突分支（DA）神经元的神经元形态的研究通过免疫荧光在神经细胞和表皮蛋白质的原位可视化中受益。我们描述通过从幼虫体壁除去肌肉组织提高多巴胺神经元和包围的表皮细胞的免疫荧光分析的过程。 

视频: Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

在活动区域​​的解剖和影像果蝇神经肌肉交界处

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

科研

神经科学

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 ...

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and mechanobiology, which all influence muscle development and myofibrillogenesis. Omics data, such as those generated by mass spectrometry or deep sequencing, can provide important mechanistic insights into these biological processes. For such approaches, it is beneficial to analyze tissue-specific samples to increase both selectivity and specificity of the omics fingerprints. Here we present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched muscle samples for omics applications. We first describe how to dissect flight muscles at early pupal stages (&#60;48 h after puparium formation [APF]), when the muscles are discernable by green fluorescent protein (GFP) labeling. We then describe how to dissect muscles from late pupae (&#62;48 h APF) or adults, when muscles are distinguishable under a dissecting microscope. The accompanying video protocol will make these technically demanding dissections more widely accessible to the muscle and Drosophila research communities. For RNA applications, we assay the quantity and quality of RNA that can be isolated at different time points and with different approaches. We further show that Bruno1 (Bru1) is necessary for a temporal shift in myosin heavy chain (Mhc) splicing, demonstrating that dissected muscles can be used for mRNA-Seq, mass spectrometry, and reverse transcription polymerase chain reaction (RT-PCR) applications. This dissection protocol will help promote tissue-specific omics analyses and can be generally applied to study multiple biological aspects of myogenesis.

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study transcriptional regulation, alternative splicing, metabolism, and mechanobiology. We present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched samples ideal for proteomics and deep-sequencing. These samples can offer important mechanistic insights into diverse aspects of muscle development.

解剖果蝇黑色素组飞行肌肉为奥米克斯方法

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and ...

遗传学

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

果蝇幼虫NMJ夹层

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

生物学

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

果蝇幼虫NMJ免疫

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

观察 果蝇 神经肌肉接头和肌肉细胞中的微管网络

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of &#945;-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

作者聚焦:了解果蝇神经肌肉接头中的微管网络 

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

使用 果蝇 幼虫神经肌肉接头和肌肉细胞可视化微管网络

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with ...

果蝇单神经元消融研究神经系统适应

Laser Cell Ablation in Intact Drosophila Larvae Reveals Synaptic Competition

The protocol describes single-neuron ablation with a 2-photon laser system in the central nervous system (CNS) of intact Drosophila melanogaster larvae. Using this non-invasive method, the developing nervous system can be manipulated in a cell-specific manner. Disrupting the development of individual neurons in a network can be used to study how the nervous system can compensate for the loss of synaptic input. Individual neurons were specifically ablated in the giant fiber system of Drosophila, with a focus on two neurons: the presynaptic giant fiber (GF) and the postsynaptic tergotrochanteral motor neuron (TTMn). The GF synapses with the ipsilateral TTMn, which is crucial to the escape response. Ablating one of the GFs in the 3rd instar brain, just after the GF starts axonal growth, permanently removes the cell during the development of the CNS. The remaining GF reacts to the absent neighbor and forms an ectopic synaptic terminal to the contralateral TTMn. This atypical, bilaterally symmetric terminal innervates both TTMns, as demonstrated by dye coupling, and drives both motor neurons, as demonstrated by electrophysiological assays. In summary, the ablation of a single interneuron demonstrates synaptic competition between a bilateral pair of neurons that can compensate for the loss of one neuron and restore normal responses to the escape circuit.

作者聚焦:研究果蝇神经回路组装和突触形成的机制

This protocol demonstrates the laser cell ablation of individual neurons in intact Drosophila larvae. The method enables the study of the effect of reducing competition between neurons in the developing nervous system.

完整 果蝇 幼虫中的激光细胞消融揭示了突触竞争

The protocol describes single-neuron ablation with a 2-photon laser system in the central nervous system (CNS) of intact Drosophila melanogaster larvae. ...

去除

摘要

探索更多视频

在活动区域的解剖和影像果蝇神经肌肉交界处

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

解剖果蝇黑色素组飞行肌肉为奥米克斯方法

果蝇幼虫NMJ夹层

果蝇幼虫NMJ免疫

石蜡包埋及冷冻的部分果蝇成人肌肉

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络

完整果蝇幼虫中的激光细胞消融揭示了突触竞争

去除

摘要

探索更多视频

在活动区域​​的解剖和影像果蝇神经肌肉交界处

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

解剖果蝇黑色素组飞行肌肉为奥米克斯方法

果蝇幼虫NMJ夹层

果蝇幼虫NMJ免疫

石蜡包埋及冷冻的部分果蝇成人肌肉

使用 果蝇 幼虫神经肌肉接头和肌肉细胞可视化微管网络

使用 果蝇 幼虫神经肌肉接头和肌肉细胞可视化微管网络

使用 果蝇 幼虫神经肌肉接头和肌肉细胞可视化微管网络

完整 果蝇 幼虫中的激光细胞消融揭示了突触竞争

在活动区域的解剖和影像果蝇神经肌肉交界处

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络

完整果蝇幼虫中的激光细胞消融揭示了突触竞争