Name: a simple one-step dissection protocol for whole-mount preparation of adult drosophila brains
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1.解决方案用于脑解剖和免疫荧光染色<ol><li>解剖成人飞脑在人造脑脊髓液（ACSF）:119 mM氯化钠，26.2毫碳酸氢钠 ，2.5mM的氯化钾，1mM的的NaH 2 PO 4，1.3毫氯化镁 ，和10mM葡萄糖。在使用前，气体用5％ 的 CO 2/95％O 2，10的脑脊液- 15分钟，穗带的2.5mM的CaCl 2。通过一个0.22微米的膜过滤器灭菌脑脊液溶液。 注:储存脑脊液在4℃，以防止细菌污染。飞大脑也可以在磷酸盐缓冲盐水（PBS）溶液解剖，虽然对解剖大脑的最终成像质量两个夹层溶液的作用详细的比较没有被执行。 </li><li>使用在1×PBS中制备的固定剂溶液，其通常等分并储存在-20℃的4％多聚甲醛（PFA）。 CautioN:PFA是有毒和腐蚀性，应小心处理。 </li><li>使用1X PBS从大脑和最后的清洗步骤后冲洗PFA，解剖大脑的免疫荧光染色过程中。从10倍的PBS原液制备1×PBS中（1.37 M氯化钠，27毫米氯化钾，100mM的磷酸氢二钠，和18毫KH 2 PO 4）。 </li><li>解剖脑的免疫荧光染色期间在1×PBS中（1×PBT）的所有随后的洗涤步骤使用0.3％吐温-20。 </li></ol> 2.成年果蝇首脑显微解剖 注意:解剖过程在图1中示出。 <ol><li>用CO 2麻醉成年果蝇。 5秒 - 使用镊子，拿起一只苍蝇，并简要由70％乙醇浸泡3就其脱蜡角质层。这确保了沉浸在脑脊液或1X PBS夹层溶液时没有气泡附着于飞角质层。 </li><li>下在那个不会被手被阻塞的角度解剖与光源的显微镜，设置物镜达到飞（1.2X倍率）一个明确的说法。使用镊子用非优势手来固定动物和浸入飞入其腹部朝上， 如图1A中的冷夹层溶液。 </li><li>保持镊子在160 - 170°角相对于夹层板，同时施加一个小的力到飞行的腹部（示出为图1A中的箭头）。这一步将固定飞行，并迫使它由15向后移动头部 - 向上延伸长鼻25°一会儿。在此过程中，表皮的柔软和半透明区域，长鼻下方，应当是显而易见的。增加放大倍率和调整焦平面到这一地区。 注意:不要拿着镊子得太紧，而只是足够牢固，能够稳定飞。用力过猛将导致内部器官破裂到溶液中。 </li><li>使用优势手按解剖镊，以便其尖端封闭的，这将产生对钳子的前端温和耐力，以便从保持手压被释放时的镊子将弹开。钳子垂直定位到保持飞镊子， 如图1B所示 。 <ol><li>皮尔斯解剖钳通过长鼻下方的角质层柔软和半透明区域闭合的提示，由图1B中的红色箭头所示。不穿透钳子太深它接触大脑是重要的。大脑正确的应该变得可见。保持飞与非优势手稳定的把握。 </li></ol></li><li>很快但稳定，靠自身的力量释放解剖钳提示，并依靠势头从这个版本到撕去Ť他的外骨骼周围的苍蝇头。一个完整的脑正确应该成为可见（白色组织立即在图1C的底部钳子的前端以上）跟随的假想线引导释放钳子提示（如在图1C中所示的向外红色箭头示出）的动能。这将温和地去除骨骼，大部分来自大脑的脑相关的眼睛和气管。适当的时机和力施加到打开外骨骼将取决于研究者的经验。 注:这是在解剖最关键的一步。它依赖于从开口钳的动量产生的自然力量，同时留下露出的脑仍附着在身体的其他部分以除去外骨骼是重要的。 </li><li>使用优势手的镊子小心地将残余附件组织，如出现白色纤维结构的气管remaini纳克附着到大脑。要小心，不要拉扯或损伤大脑正确的。 </li></ol> 3.大脑的免疫荧光染色<ol><li>固定在约1毫升4％PFA进行40与它们相关的躯干的解剖脑样品 - 60分钟。 注意:对于这个和随后的步骤，保持上一章动器的管或板的样品在溶液中适当地混合的样品。 </li><li>吹打解决方案上下3次用1毫升1X PBS的冲洗。小心不要吸出的大脑了。 </li><li>用1×PBT洗5次，每次5分钟。保持对章动器样品孵化过程中。 </li><li>一抗孵育适当的稀释O / N在4℃。 </li><li>第二天，除去第一抗体，并用1×PBT洗样品5次。离开样品5 - 在1倍的PBT溶液10分钟，每次洗涤之间。保持对章动器样品孵化过程中。 </li><li>孵育洗样中所建议的稀释和培养时间适当的二级抗体。 </li><li>用1X PBT洗样品六次每次冲洗之间的温育10分钟。 本步骤是从样品中除去非特异性结合的第二抗体是至关重要的。 </li><li>孵育以1:在1×PBT溶液的1毫克/毫升4&#39;，6&#39;-二脒基-2-苯基吲哚（DAPI）1:10,000稀释30分钟。 DAPI是结合DNA的AT区，可用于揭示脑的整体形态的DNA染料。 </li><li>用1X PBS冲洗三次。 </li><li>转移样品解剖菜，然后使用镊子解剖盘分开尸体的大脑。 </li><li>后染色和洗涤步骤，装入在盖玻片两者之间的大脑，并放置在盖玻片上的安装滑道的顶部。以这种方式，所述脑样品可以容易地翻转使大脑的两侧可具有类似的信号强度进行成像。 <strong&gt;注意:安装脑盖玻片上，而不是直接在安装滑道使得样品的方向翻转自如。安装工序是在下面更详细说明。 <ol><li>放置在安装滑道的顶部的18×18mm的盖玻片。加宽枪头用刀片的开口，并用它来在1×PBS溶液转移大脑到18×18mm的盖玻片。 </li><li>放置的大脑上盖玻片的中间，除去用细枪头大脑周围液体，然后添加另外20 - 抗褪色封固剂的40微升（0.2％（重量/体积）正丙基99.5没食子酸％ACS级甘油）在大脑。 注:固定介质中加入的量取决于研究大脑的数量。 </li><li>轻轻散开的大脑，而向外移位粘性安装介质。放置在脑的顶部的第二18×18mm的盖玻片通过首先从一侧降低滑动直到其与接触安装滑的表面上，然后慢慢降低的另一侧，以尽量减少气泡脑接触。 注意:没有必要使用垫片在两个盖玻片之间，作为安装培养基的体积和大脑应提供足够的两个盖玻片分开的空间。 </li><li>允许的幻灯片落户。除去过量的液体，从用实验室组织卡瓦两侧出来。 </li><li>为了防止安装的大脑盖玻片在运输过程中脱落的安装滑轨，用一小块胶带将盖玻片安装到固定的幻灯片。 注意:脑安装在之间的幻灯片可以立即被成像或在没有荧光信号的显著损失4℃长达一个星期保存下来，而不需要使用任何密封剂密封的幻灯片。对于染色的大脑长期保存，这两个滑动的侧可被密封瓦特第i指甲油，并存储在-20℃冰箱，虽然染色的大脑可能会失去它们的信号在一段时间。这是不推荐的。 </li></ol></li><li>使用适当的复合荧光显微镜或共聚焦显微镜图像的大脑。 <ol><li>首先，从大脑更靠近物镜的一侧获取图像，然后小心地翻转的盖玻片有夹在中间的试样。这个步骤有利于获得相同的大脑的另一侧的图像。 </li><li>使用一个直立化合物荧光显微镜和20X物镜到图像全脑（ 图2中的例子）和40X物镜到大脑的图像较小的区域，如在配对前内侧（PAM）簇区多巴胺神经元（示例在图3中）。 </li></ol></li></ol>

<ol>
	<li>Wangler, M. F., Yamamoto, S., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fruit+flies+in+biomedical+research.">Fruit flies in biomedical research.</a> Genetics. 199, 639-653 (2015).</li><li>Bellen, H. J., Yamamoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morgan's+legacy:+fruit+flies+and+the+functional+annotation+of+conserved+genes.">Morgan's legacy: fruit flies and the functional annotation of conserved genes.</a> Cell. 163, 12-14 (2015).</li><li>Aso, Y., 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+neuronal+architecture+of+the+mushroom+body+provides+a+logic+for+associative+learning.">The neuronal architecture of the mushroom body provides a logic for associative learning.</a> Elife. 3, 04577 (2014).</li><li>Reiter, L. T., Potocki, L., Chien, S., Gribskov, M., Bier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+analysis+of+human+disease-associated+gene+sequences+in+Drosophila+melanogaster.">A systematic analysis of human disease-associated gene sequences in Drosophila melanogaster.</a> Genome Res. 11, 1114-1125 (2001).</li><li>Yamagata, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+dopamine+neurons+mediate+reward+signals+for+short-+and+long-term+memories.">Distinct dopamine neurons mediate reward signals for short- and long-term memories.</a> Proc Natl Acad Sci U S A. 112, 578-583 (2015).</li><li>Nern, A., Pfeiffer, B. D., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+tools+for+multicolor+stochastic+labeling+reveal+diverse+stereotyped+cell+arrangements+in+the+fly+visual+system.">Optimized tools for multicolor stochastic labeling reveal diverse stereotyped cell arrangements in the fly visual system.</a> Proc Natl Acad Sci U S A. 112, 2967-2976 (2015).</li><li>Waddell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+Plasticity:+Dopamine+Tunes+the+Mushroom+Body+Output+Network.">Neural Plasticity: Dopamine Tunes the Mushroom Body Output Network.</a> Curr Biol. 26, 109-112 (2016).</li><li>Wolff, T., Iyer, N. A., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroarchitecture+and+neuroanatomy+of+the+Drosophila+central+complex:+A+GAL4-based+dissection+of+protocerebral+bridge+neurons+and+circuits.">Neuroarchitecture and neuroanatomy of the Drosophila central complex: A GAL4-based dissection of protocerebral bridge neurons and circuits.</a> J Comp Neurol. 523, 997-1037 (2015).</li><li>Sweeney, S. T., Hidalgo, A., de Belle, J. S., Keshishian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+adult+Drosophila+brains.">Dissection of adult Drosophila brains.</a> Cold Spring Harb Protoc. 2011, 1472-1474 (2011).</li><li>Wu, J. S., 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=A+protocol+for+dissecting+Drosophila+melanogaster+brains+for+live+imaging+or+immunostaining.">A protocol for dissecting Drosophila melanogaster brains for live imaging or immunostaining.</a> Nat Protoc. 1, 2110-2115 (2006).</li><li>Mao, Z., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eight+different+types+of+dopaminergic+neurons+innervate+the+Drosophila+mushroom+body+neuropil:+anatomical+and+physiological+heterogeneity.">Eight different types of dopaminergic neurons innervate the Drosophila mushroom body neuropil: anatomical and physiological heterogeneity.</a> Front Neural Circuits. 3, 5 (2009).</li><li>White, K. E., Humphrey, D. M., Hirth, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dopaminergic+system+in+the+aging+brain+of+Drosophila.">The dopaminergic system in the aging brain of Drosophila.</a> Front Neurosci. 4, 205 (2010).</li><li>Yang, Y., 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=Mitochondrial+pathology+and+muscle+and+dopaminergic+neuron+degeneration+caused+by+inactivation+of+Drosophila+Pink1+is+rescued+by+Parkin.">Mitochondrial pathology and muscle and dopaminergic neuron degeneration caused by inactivation of Drosophila Pink1 is rescued by Parkin.</a> Proc Natl Acad Sci U S A. 103, 10793-10798 (2006).</li><li>Greene, J. 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=Mitochondrial+pathology+and+apoptotic+muscle+degeneration+in+Drosophila+parkin+mutants.">Mitochondrial pathology and apoptotic muscle degeneration in Drosophila parkin mutants.</a> Proc Natl Acad Sci U S A. 100, 4078-4083 (2003).</li><li>Whitworth, A. 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=Increased+glutathione+S-transferase+activity+rescues+dopaminergic+neuron+loss+in+a+Drosophila+model+of+Parkinson's+disease.">Increased glutathione S-transferase activity rescues dopaminergic neuron loss in a Drosophila model of Parkinson's disease.</a> Proc Natl Acad Sci U S A. 102, 8024-8029 (2005).</li><li>Pesah, Y., 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=Drosophila+parkin+mutants+have+decreased+mass+and+cell+size+and+increased+sensitivity+to+oxygen+radical+stress.">Drosophila parkin mutants have decreased mass and cell size and increased sensitivity to oxygen radical stress.</a> Development. 131, 2183-2194 (2004).</li><li>Trinh, 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=Decaffeinated+coffee+and+nicotine-free+tobacco+provide+neuroprotection+in+Drosophila+models+of+Parkinson's+disease+through+an+NRF2-dependent+mechanism.">Decaffeinated coffee and nicotine-free tobacco provide neuroprotection in Drosophila models of Parkinson's disease through an NRF2-dependent mechanism.</a> J Neurosci. 30, 5525-5532 (2010).</li><li>Kim, K., Kim, S. H., Kim, J., Kim, H., Yim, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutathione+s-transferase+omega+1+activity+is+sufficient+to+suppress+neurodegeneration+in+a+Drosophila+model+of+Parkinson+disease.">Glutathione s-transferase omega 1 activity is sufficient to suppress neurodegeneration in a Drosophila model of Parkinson disease.</a> J Biol Chem. 287, 6628-6641 (2012).</li></ol>

The authors have nothing to disclose.

在使用成年果蝇脑研究人类脑疾病，神经元的电路，和更高的脑功能的兴趣日益增加，需要开发简单和快速的方法，以获得完整的飞大脑对于整个贴装分析，这是适用于大尤其重要比例基于大脑的屏幕。我们的方法提供了一个简单，易于学习的方法来剖析出一只苍蝇头（通常在不到10秒的经验）有保存完好的形态被清除大部分相关的组织。作为解剖大脑仍附着到飞体的其余部分，它们通常?...

模式生物果蝇 ，俗称实蝇，长期以来一直重视其优雅的基因工具，短生育时间和高度保守的分子和细胞通路。果蝇已成功地用于解剖基本信号通路，多细胞生物体的图案化的机制，以及神经元发育，功能的基本机制，和疾病1,2-。在细胞标记和成像技术的最新进展，果蝇大脑已经成为神经回路的精细定位，并在解剖的高级脑功能，例如学习，记忆，和昼夜节律1,3的分子和细胞基础特别强大， 4,5,6,7,8。 果蝇系统的一个特别的优点是它的相对小的尺寸，允许使用常规化合物或共焦显微镜的大脑整个安装制备和检查。氏取值特征使神经元电路的详细解剖和功能分析，或甚至一个单一的神经元，在细胞和亚细胞水平，在全脑组织的范围内，从而在整个内提供所研究的受试者的两个整体视图及其精确几何脑。然而，考虑到大脑的相当小尺寸，它也呈现在一个成年果蝇有效解剖一个完整的脑组织从保护外骨骼头壳体的技术挑战。各种有效和相对简单的解剖的方法已详细描述，其通常涉及仔细和分步除去头部壳体的和相关的组织，包括眼睛，气管，和脂肪从大脑适当9,10，这些显微解剖的方法经常放置在解剖钳的质量相当严格的要求，依托细以及对齐提示，可以很容易损坏钳。此外，作为解剖大脑往往分隔符从身体的其余编，大脑可以容易地期间，因为它们的小尺寸和它们在处理缓冲器透明度的后续染色和洗涤过程丢失。在这里，我们描述了，保持连接到躯干的解剖大脑成人大脑相对简单和容易学习，一步夹层协议。解剖过程中往往容易清除了大部分脑相关组织如眼睛和气管，并减少了质量好的解剖钳的需求。 此外，荧光化合物显微镜或共焦显微镜下成像大脑时，大脑的一侧即来自荧光光源远经常产生一个较弱的信号和较少的清晰图像，由于整个贴装脑的厚度。在这里，我们还描述了一个简单的安装方法，其允许大脑样品容易翻转，使大脑两侧方便成像相似的信号的IntensiTY和质量。 作为证明的概念对于本方法的应用，研究成人脑，我们进一步研究多巴胺神经元的用w 1118蝇脑中的存在;被经常用作用于产生转基因苍蝇，在许多果蝇研究野生型对照的亲本系基因型。

<table><tbody><tr><td>w&lt;sup&gt;*&lt;/sup&gt;;&amp;nbsp;park&lt;sup&gt;&amp;Delta;21&lt;/sup&gt;/TM3,&amp;nbsp;P{GAL4-Kr.C}DC2,&amp;nbsp;P{UAS-GFP.S65T}DC10,&amp;nbsp;Sb&lt;sup&gt;1&lt;/sup&gt;</td><td>Bloomington Drosophila Stock Center</td><td>51652</td><td>Balancer was switched to TM6B</td></tr><tr><td>PBac{WH}park&lt;sup&gt;f01950&lt;/sup&gt;</td><td>Exelixis at Harvard Medical School</td><td>f01950</td><td>Balancer was switched to TM6C</td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>S640-500</td><td></td></tr><tr><td>Sodium Bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)&amp;nbsp;</td><td>Fisher Scientific</td><td>02-003-990</td><td></td></tr><tr><td>Potassium Chloride (KCl)</td><td>Fisher Scientific</td><td>BP366-500</td><td></td></tr><tr><td>Sodium phosphate, monobasic monohydrate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>02-004-198</td><td></td></tr><tr><td>Magnesium Chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>02-003-265</td><td></td></tr><tr><td>D-Sorbitol</td><td>Sigma-Aldrich</td><td>S1876-500G</td><td>Replaces glucose</td></tr><tr><td>Calcium chloride dihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>C5670-500G</td><td></td></tr><tr><td>EMD Millipore&amp;nbsp;Durapore PVDF Membrane Filters: Hydrophilic: 0.22 &amp;micro;m Pore Size</td><td>Fisher Scientific</td><td>GVWP14250</td><td></td></tr><tr><td>Formalin Solution, 10% (Histological)</td><td>Fisher Scientific</td><td>SF98-20</td><td></td></tr><tr><td>Potassium Phosphate, Dibasic, Powder, Ultrapure Bioreagent</td><td>Fisher Scientific</td><td>02-003-823</td><td></td></tr><tr><td>Tween-20</td><td>Fisher Scientific</td><td>BP337-500</td><td></td></tr><tr><td>Excelta Precision Tweezers with Very Fine Points</td><td>Fisher Scientific</td><td>17-456-055</td><td>Protocol does not require very fine points.&amp;nbsp;</td></tr><tr><td>Anti-Tyrosine Hydroxylase Antibody</td><td>Pel-Freez Biologicals</td><td>P40101</td><td></td></tr><tr><td>Rat-Elav-7E8A10 anti-elav</td><td>The Developmental Studies Hybridoma Bank</td><td>Clone 7E8A10</td><td></td></tr><tr><td>Goat anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 647 conjugate</td><td>ThermoFisher Scientific</td><td>A-21247</td><td></td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 594 conjugate</td><td>ThermoFisher Scientific</td><td>A-11037</td><td></td></tr><tr><td>DAPI Solution (1 mg/mL)</td><td>ThermoFisher Scientific</td><td>62248</td><td></td></tr><tr><td>Propyl gallate powder</td><td>Sigma-Aldrich</td><td>P3130-100G</td><td></td></tr><tr><td>Glycerol ACS reagent, &amp;ge;99.5%</td><td>Sigma-Aldrich</td><td>G7893-500ML</td><td></td></tr><tr><td>Zeiss Axioimager Z1</td><td>Zeiss</td><td>Quote</td><td></td></tr><tr><td>Zeiss Apotome.2</td><td>Zeiss</td><td>Quote</td><td></td></tr><tr><td>Zen lite software</td><td></td><td>Quote</td><td></td></tr></tbody></table>

a simple one-step dissection protocol for whole-mount preparation of adult drosophila brains

有在使用果蝇到人脑退行性疾病的模型，在成人大脑映射神经设计电路，和研究的高级脑功能的分子和细胞基础的兴趣日益增加。成人大脑的整体卡口制剂保存完好的形态是这样的全脑为基础的研究关键的，但也可以是技术挑战性和耗时。这个协议描述了成年果蝇头部在不到10秒的一个易于了解，一步法解剖的方法，同时保持附连到身体的其他部分的完整的脑，以方便后续处理步骤。该过程有助于消除最通常与可以与稍后成像步骤干涉大脑相关的眼睛和气管组织的，并且也对所述解剖钳的质量需求较少。此外，我们描述一个简单的方法，可用于在盖玻片安装的脑样品的方便翻转，这是用于成像的B的两侧重要降雨具有类似信号强度和质量。作为协议的一个例子，我们在野生成年大脑多巴胺能呈现（DA）神经元的分析（ 附1118）苍蝇。解剖方法的疗效高，使其成为在果蝇中大型为主脑的成人研究特别有用。

图1示出了用于成人脑解剖主程序中，如上所述图2和3是3日龄WT代表性图像（基因型:W 1118）成人飞大脑，其被costained用抗酪氨酸羟化酶的抗体（TH ，用红色的图2和白色图3），一个标记通常用于标记多巴胺神经元11中，除了将DNA染料DAPI标记所有所有分化的神经元细胞的细胞核和针对ELAV蛋...

Watch this Scientific Journal Video about A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains at JoVE.com

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

成年果蝇大脑是研究神经回路，大脑高级功能和复杂性疾病的宝贵制度。一种有效的方法来剖析来自小苍蝇头全脑组织将促进脑为基础的研究。在这里，我们描述了一个简单的一步解剖成人的大脑保存完好的形态协议。

一个简单的一步解剖协议成人全安装准备<em&gt;果蝇</em&gt;脑

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

a-simple-one-step-dissection-protocol-for-whole-mount-preparation

Research

JoVE Journal

Neuroscience

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

27.1K 次观看.  The University of Texas Health Science Center at Houston. 成年果蝇大脑是研究神经回路，大脑高级功能和复杂性疾病的宝贵制度。一种有效的方法来剖析来自小苍蝇头全脑组织将促进脑为基础的研究。在这里，我们描述了一个简单的一步解剖成人的大脑保存完好的形态协议。 

视频: A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

Cells display different morphologies and complex anatomical relationships. How do cells interact with their neighbors? Do the interactions differ between cell types or even within a given type? What kinds of spatial rules do they follow? The answers to such fundamental questions in vivo have been hampered so far by a lack of tools for high resolution single cell labeling. Here, a detailed protocol to target single cells with a MultiColor FlpOut (MCFO) technique is provided. This method relies on three differently tagged reporters (HA, FLAG and V5) under UAS control that are kept silent by a transcriptional terminator flanked by two FRT sites (FRT-stop-FRT). A heat shock pulse induces the expression of a heat shock-induced Flp recombinase, which randomly removes the FRT-stop-FRT cassettes in individual cells: expression occurs only in cells that also express a GAL4 driver. This leads to an array of differently colored cells of a given cell type that allows the visualization of individual cell morphologies at high resolution. As an example, the MCFO technique can be combined with specific glial GAL4 drivers to visualize the morphologies of the different glial subtypes in the adult Drosophila brain.

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

Cells display different morphologies and establish a variety of interactions with their neighbors. This protocol describes how to reveal the morphology of single cells and to investigate cell-cell interaction by using the well-established Gal4/UAS expression system.

多色 FlpOut 技术在果蝇中的应用研究胶质细胞的高分辨率单细胞形态学

Cells display different morphologies and complex anatomical relationships. How do cells interact with their neighbors? Do the interactions differ between ...

科研

行为学

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

发育生物学

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

发展和成人的制备果蝇大脑和视网膜实时成像

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

生物学

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the developmental mechanisms that generate neural diversity and drive circuit assembly. Given its complex three-dimensional organization, analysis of the optic lobe requires that one understand how its adult neuropils and larval progenitors are positioned relative to each other and the central brain. Here, we describe a protocol for the dissection, immunostaining and mounting of larval and adult brains for optic lobe imaging. Special emphasis is placed on the relationship between mounting orientation and the spatial organization of the optic lobe. We describe three mounting strategies in the larva (anterior, posterior and lateral) and three in the adult (anterior, posterior and horizontal), each of which provide an ideal imaging angle for a distinct optic lobe structure.

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

This protocol describes three steps to prepare larval and adult Drosophila optic lobes for imaging: 1) brain dissections, 2) immunohistochemistry and 3) mounting. Emphasis is placed on step 3, as distinct mounting orientations are required to visualize specific optic lobe structures.&#160;

解剖、免疫组织化学和幼虫和成年 果蝇 脑的安装用于视叶可视化

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the ...

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our understanding of the molecular mechanisms underlying axon degeneration remains limited. Injury-induced axon degeneration serves as a simple model to study how severed axons execute their own disassembly (axon death). Over recent years, an evolutionarily conserved axon death signaling cascade has been identified from flies to mammals, which is required for the separated axon to degenerate after injury. Conversely, attenuated axon death signaling results in morphological and functional preservation of severed axons and their synapses. Here, we present three simple and recently developed protocols that allow for the observation of axonal morphology, or axonal and synaptic function of severed axons that have been cut-off from the neuronal cell body, in the fruit fly Drosophila. Morphology can be observed in the wing, where a partial injury results in axon death side-by-side of uninjured control axons within the same nerve bundle. Alternatively, axonal morphology can also be observed in the brain, where the whole nerve bundle undergoes axon death triggered by antennal ablation. Functional preservation of severed axons and their synapses can be assessed by a simple optogenetic approach coupled with a post-synaptic grooming behavior. We present examples using a highwire loss-of-function mutation and by over-expressing dnmnat, both capable of delaying axon death for weeks to months. Importantly, these protocols can be used beyond injury; they facilitate the characterization of neuronal maintenance factors, axonal transport, and axonal mitochondria.

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Here, we provide protocols to perform three simple injury-induced axon degeneration (axon death) assays in Drosophila melanogaster to evaluate the morphological and functional preservation of severed axons and their synapses.

在果蝇黑色无gaster的Axon死亡期间，对Axon及其突触的形态和功能评估

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our ...

神经科学

Direct Cryosectioning of Drosophila Heads for Enhanced Brain Fluorescence Staining and Immunostaining

Immunostaining Drosophila melanogaster brains is essential for exploring the mechanisms behind complex behaviors, neural circuits, and protein expression patterns. Traditional methods often involve challenges such as performing complex dissection, maintaining tissue integrity, and visualizing specific expression patterns during high-resolution imaging. We present an optimized protocol that combines cryosectioning with fluorescence staining and immunostaining. This method improves tissue preservation and signal clarity and reduces the need for laborious dissection for Drosophila brain imaging. The method entails rapid dissection, optimal fixation, cryoprotection, and cryosectioning, followed by fluorescent staining and immunostaining. The protocol significantly reduces tissue damage, enhances antibody penetration, and yields sharp, well-defined images. We demonstrate the effectiveness of this approach by visualizing specific neural populations and synaptic proteins with high fidelity. This versatile method allows for the analysis of various protein markers in the adult brain across multiple z-planes and can be adapted for other tissues and model organisms. The protocol provides a reliable and efficient tool for researchers conducting high-quality immunohistochemistry in Drosophila neurobiology studies. This method's detailed visualization facilitates comprehensive analysis of neuroanatomy, pathology, and protein localization, making it particularly valuable for neuroscience research.

This study presents a simplified protocol for tissue processing involving decapitation, fixation, cryosectioning, fluorescence staining, immunostaining, and imaging, which can be extended to confocal and multiphoton imaging. The method maintains efficacy comparable to complex dissections, bypassing the need for advanced motor skills. Quantitative image analysis provides extensive investigative potential.

果 蝇 头部的直接冷冻切片，用于增强脑荧光染色和免疫染色

Immunostaining Drosophila melanogaster brains is essential for exploring the mechanisms behind complex behaviors, neural circuits, and protein expression ...

Mounting Adult Drosophila Brains: A Method to Prepare Slides for Confocal Imaging

Source: Kelly, S. M., et al. <a href="https://www.jove.com/video/56174/" target="_blank">Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains</a>. J. Vis. Exp. (2017).
This video describes a method of mounting intact brains from adult flies under a bridge slide and shows the protocol performed on immunostained brains for fluorescent microscopy.

安装成体果蝇大脑:一种为共聚焦成像准备载玻片的方法

Source: Kelly, S. M., et al. Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains. ...

JoVE Encyclopedia of Experiments

黑腹果蝇（果蝇）

成虫

Drosophila Adult Brain Dissection: A Method in Fly Neurobiology

Source: Kelly, S. M., et al. <a href="https://www.jove.com/video/56174/" target="_blank">Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains</a>. J. Vis. Exp. (2017).
This video describes how to dissect and isolate the adult Drosophila brain&#8211;a procedure necessary for visualizing the organ, which is otherwise obscured by the cuticle, eye, and tracheal tissue. The example protocol shows a detailed demonstration yielding high-quality preparations that can be used for immunostaining and imaging methods in Drosophila neurobiology.

果蝇成体脑解剖:果蝇神经生物学中的一种方法

Imaging and Quantification of Protein Aggregates in Genetically Modified Drosophila Brains

Source: Donnelly, K. M., et al. <a href="https://app.jove.com/v/56906">Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster.</a> J. Vis. Exp. (2018)
This video demonstrates imaging and quantification of protein aggregates in genetically modified Drosophila brains. Brains are fixed, washed, and treated with an antifading agent before mounting. Images are captured using a confocal microscope and quantified to observe the transmission of mutant protein aggregates.

转基因果蝇大脑中蛋白质聚集体的成像和定量

1. Coupling Gal4- and QF-mediated Htt Transgene Expression in Drosophila

	Collect and/or generate transgenic Drosophila melanogaster lines containing ...

Neuropathology

Neurodegenerative Diseases

Immunofluorescence Staining of Drosophila Brains for the Single-Cell Imaging of Glial Cells

Source: Batelli, S., et al. <a href="https://app.jove.com/v/56177">Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila.</a>&#160;J. Vis. Exp. (2017).
This video demonstrates the immunofluorescence staining of a transgenic Drosophila brain for single-cell imaging of glial cells. The brain expresses membrane-targeted proteins fused with different combinations of antigenic epitopes under the control of a glial-specific recombinase system. The isolated and fixed brains are stained with primary antibodies specific to the antigenic epitopes, followed by fluorophore-conjugated secondary antibodies. This labeling enables the simultaneous imaging of different glial cell subpopulations across various brain regions.

Source: Batelli, S., et al. Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in ...

一个简单的一步解剖协议成人全安装准备

摘要

探索更多视频

此视频中的章节

多色 FlpOut 技术在果蝇中的应用研究胶质细胞的高分辨率单细胞形态学

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

发展和成人的制备果蝇大脑和视网膜实时成像

解剖、免疫组织化学和幼虫和成年果蝇脑的安装用于视叶可视化

在果蝇黑色无gaster的Axon死亡期间，对Axon及其突触的形态和功能评估

果蝇头部的直接冷冻切片，用于增强脑荧光染色和免疫染色

安装成体果蝇大脑:一种为共聚焦成像准备载玻片的方法

果蝇成体脑解剖:果蝇神经生物学中的一种方法

转基因果蝇大脑中蛋白质聚集体的成像和定量

Immunofluorescence Staining of Drosophila Brains for the Single-Cell Imaging of Glial Cells

一个简单的一步解剖协议成人全安装准备

摘要

探索更多视频

此视频中的章节

多色 FlpOut 技术在果蝇中的应用研究胶质细胞的高分辨率单细胞形态学

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

发展和成人的制备果蝇大脑和视网膜实时成像

解剖、免疫组织化学和幼虫和成年 果蝇 脑的安装用于视叶可视化

在果蝇黑色无gaster的Axon死亡期间，对Axon及其突触的形态和功能评估

果 蝇 头部的直接冷冻切片，用于增强脑荧光染色和免疫染色

安装成体果蝇大脑:一种为共聚焦成像准备载玻片的方法

果蝇成体脑解剖:果蝇神经生物学中的一种方法

转基因果蝇大脑中蛋白质聚集体的成像和定量

Immunofluorescence Staining of Drosophila Brains for the Single-Cell Imaging of Glial Cells

解剖、免疫组织化学和幼虫和成年果蝇脑的安装用于视叶可视化

果蝇头部的直接冷冻切片，用于增强脑荧光染色和免疫染色