Name: application of multicolor flpout technique to study high resolution single cell morphologies and cell interactions of glia in drosophila
Uploaded: 2017-10-20T14:00:00.000+00:00
Duration: 8 min 30 s
Description: Watch this Scientific Journal Video about Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila at JoVE.com

作者感谢 Arnim Jenett, Aljoscha 海滨, 和鲁宾实验室的其他成员的建议和共享的未发表的试剂和 Janelia 飞光项目组产生共焦图像。作者还感谢高卢实验室的成员对手稿的评论。

1. 为多色 FlpOut (MCFO) 实验准备苍蝇 注意: MCFO 技术指的是所谓的 Flp 介导的停止盒式切除 (FlpOut) 的修改版本。转基因 MCFO 蝇携带热休克启动子 (hsp)-Flp 重组和不同的记者在无人控制下。每个记者都由一个 myristoylated (myr) 超级文件夹绿色荧光蛋白 (sfGFP) 的共同骨干组成, 其中10个表位标签 ( e. g ., HA, 旗或 V5) 的副本已经插入。所产生的荧光蛋白的命名和 #34; 意大利面条怪兽协调和 #34; (smGFPs) 14 , 可以使用针对不同表位标记的特定抗体检测到. 
<ol> <li> 与 MCFO 磁带和 hsp Flp (hsp-Flp; 10 xuas-首次登记税-停止-首次登记税-myr-smGFP-房委会, 10 xuas-首次登记税-停止-首次登记税-myr-smGFP-旗帜, 10 xuas-首次登记-停止-首次登记-myr-smGFP-V5) 到适当的神经胶质 GAL4 驱动程序线.
	<ol> <li> 由于 hsp 在25和 #176 已处于活动状态; c 和少数细胞可能会进行重组, 导致不标记, 设置十字在18和 #176; c, 以避免系统泄漏。在18和 #176 等待大约20天; C 要获得 F1 生成。在热冲击以后 (参见步骤 2), 维护飞行在25和 #176; C ( 图 3 ). </li>
	</ol> </li> </ol> 2。热冲击 注意: 根据插入点的不同, Flp 的效率可能会有所不同, 因此, 最佳的热冲击时间必须优化。对于这个协议, 在 X 染色体上插入热休克蛋白-Flp 的 MCFO 飞行储存 (参见 材料表 ). 
<ol> <li> 在步骤 1.1 (F1 代蝇、3-4 天) 中将十字的子代转移到包含食品和热休克的新瓶中37和 #176; C 在水浴. 
	注: 幼蝇 (1-2 天) 对热休克非常敏感。等待1或2天以上的热休克, 以降低死亡率的治疗造成的。使用一些 F1 代苍蝇作为一个控制, 没有热休克. </li>
	<li> 在热休克期间, 在正常的小瓶中维持苍蝇的食物, 以降低压力导致的死亡率。把雌性和雄性放在不同的瓶子里. </li>
	<li> 请务必将整个瓶子浸入水中, 以确保均匀的热休克。使用5-8 分钟休克作为一个适当的出发点, 稀疏标签的胶质细胞与许多 GAL4 驱动线;冲击持续时间必须优化每一个驱动程序和实验 (更短或更长的热休克时间可能是必要的). </li>
	<li> 热休克后, 将小瓶水平放置在长凳上几分钟, 以便让苍蝇从热休克中恢复. 
	注: 不需要更换药瓶. </li>
	<li> 保持25和 #176 的苍蝇; C 和解剖 (见步骤3和 4) 他们2或更多天后, 热休克的最佳记者表达. </li>
</ol> 3。解剖准备和解决方案 <ol> <li> 解剖的当天, 在200和 #181 中准备新的固定液; 通过混合180和 #181 的 S2 细胞培养基, 以20和 #181; l. 20% 甲醛 (粉煤灰), 以获得2% 的粉煤灰溶液。在解剖之前和期间保持固定液在冰上. 
	注意: 粉煤灰是有毒的, 必须小心处理. 
	注: 混合160和 #181; l S2 细胞培养基与40和 #181; l 20% 甲醛 (粉煤灰) 在200和 #181; l PCR 管制备4% 的煤灰溶液而不是2% 的溶液. </li>
	<li> 使用一个 PCR 管每个基因型与最多8-10 大脑每管最佳染色结果. </li>
	<li> 准备成人大脑洗涤解决方案: 0.5% 牛血清白蛋白, 0.5% X-100 在 PBS. 
	注: 根据染色, 可能需要一个包含 1% X-100 的溶液。更高浓度的海卫一 X-100 增加了抗体的渗透到组织和它是必要的染色区域深处的组织 ( 例如, 突触区域在成人的 果蝇 脑). </li>
	<li> 准备堵塞解决方案: 3% 正常山羊血清, 3% 正常驴血清, 0.5% X-100 在 PBS. </li>
	<li> 成人洗脑液和阻断溶液可存储在4和 #176; C 几个星期. </li>
	<li> 将深凹陷井玻璃板放在冰上, 并用以下溶液填充, 其中有70% 乙醇, 一个带有 PBS, 一个带有 S2 细胞培养基. </li>
</ol> 4。成人大脑解剖 <ol> <li> 在显微镜的舞台上放置一个3厘米的解剖盘, 并用冷 S2 细胞培养基填充。在这个培养基中进行所有的解剖, 以确保组织在解剖过程中保持健康. 
	注: 使用解剖盘内衬几毫米黑色硅胶提供了良好的背景对比 (在图像), 并保存在解剖钳. </li>
	<li> 麻醉所需基因型的苍蝇与 CO 2 . </li>
	<li> 用镊子, 抓住苍蝇的翅膀和洗涤它在冷70% 乙醇三十年代, 然后与冷 PBS 额外三十年代. </li>
	<li> 将苍蝇移入深凹陷, 充满 S2 细胞培养基. </li>
	<li> 对相同基因型的所有果蝇重复相同的过程, 并在冷 S2 细胞培养基中保持它们直到解剖, 这将保存组织. 
	注: 麻醉在 S2 细胞培养基中, 只有在30分钟长潜伏期的时间内可以解剖的苍蝇数量可能会损伤组织. </li>
	<li> 用镊子抓苍蝇, 在显微镜下, 将苍蝇浸入 S2 细胞培养基中. </li>
	<li> 将头部与身体其他部分分离。丢弃身体, 将头部浸入 S2 细胞培养基中。在整个解剖过程中, 用钳子钳住头部是极其重要的。如果头部开始漂浮, 就很难在不损坏组织的情况下进行检索. </li>
	<li> 通过拉出一只眼睛开始解剖, 同时始终保持头部至少有一个镊子。确保镊子的尖端就在视网膜下面, 以避免破坏下面的组织。拉出另一只眼睛, 开始去除角质层, 直到大脑出现. </li>
	<li> 将所有气管组织和角质层移到大脑周围, 直到组织出现清洁。确保将所有的气管组织移到大脑周围, 以获得最佳的染色效果;现在, 这些组织已经准备好被修复和染色. </li>
	<li> 在冷 S2 细胞培养基中保持所有解剖的脑部, 然后将其转移到粉煤灰溶液中, 以便时间固定. </li>
</ol> 5。成人脑部染色 ol> 在室温 (RT) 中, 用 P10 吸管将分离的大脑转移到粉煤灰溶液中。为了避免组织损伤, 永远不要用镊子在解剖后转移大脑。在200和 #181 中执行所有步骤, 在 nutator 上进行 PCR. </li>
	<li> 对于所有步骤, 在用 P200 吸管丢弃旧的解决方案之前, 允许大脑在管的底部进行沉淀。试着除去上清液而不吸进组织。保护组织免受光照射. </li>
	<li> 在200和 #181 中修复大脑; 2% 粉煤灰在 S2 细胞培养基中为1小时或4% 粉煤灰30分钟。在样品间保持固定时间相同, 以提高重现性. </li>
	<li> 3 (或更多) 洗涤15分钟, 每200和 #181; 成人脑冲洗液, 用200和 #181 阻断组织; l 阻塞溶液30分钟。更长的潜伏期在阻拦解答是可能的. </li>
	<li> 在4和 #176 中一夜之间孵化大脑; C. 在成人脑洗涤液中稀释的初级抗体, 总容积为200和 #181; L. 
	注意: 潜伏期可能会因所使用的抗体类型而异。可能需要更长的孵化。
	<ol> <li> 用于标记三 MCFO 标记, 用反 HA (1:500)、反旗 (DYKDDDDK 表位) (1:100) 和 anti-V5 主抗体孵育大脑. </li>
		<li> 主直接共轭抗体可用于替代正常的主 + 次组合 ( e. g ., anti-V5:DyLight 549 (1:200))。在这种情况下, 将直接共轭抗体视为二次抗体. 
		注意: 对于每个基因型, 保持一些大脑作为对照, 以验证染色的特异性。跳过主抗体培养, 并直接进入下一步. </li>
	</ol> </li> <li> 在200和 #181 中为1小时清洗组织3次; 成人洗脑液 (步骤 3.3), 并在成人脑洗涤液中用二次荧光-共轭/原直接共轭抗体进行孵育, 在晚上4#176; C 或在 RT 上为4小时, 总容量为200和 #181; L 注意: 适当的抗体的例子: AlexaFluor 488 (1:250), anti-V5:DyLight 549 (1:200) 和 DyLight 647 (1:100)-共轭抗体. </li>
	<li> 在200和 #181 中洗脑3次1小时; 成人洗脑液, 在200和 #181 的最后一次洗涤, 在4和 #176; C 或1-2 小时; 在 pbs 的最后洗涤步骤删除任何痕迹的海卫一 X-100, 是必要的最佳染色效果。装在安装介质上的大脑, 在玻璃片上有一个防剂. </li>
</ol> 6。安装的大脑成像 <ol> <li> 使用剪刀将成像间隔剪裁到适当的大小。对于高分辨率显微镜, 三明治的标本和成像间隔两个玻璃片. 
	注: 成像垫片是薄 (0.12 毫米厚) 胶粘剂垫片, 剥离和坚持玻璃片或显微镜幻灯片, 允许安装的标本没有压缩. </li>
	<li> 使用镊子, 从一个表面上取下粘合剂衬垫, 然后在玻璃片 (22 mm x 60 mm) 的表面上应用垫片、胶粘剂侧。应用10和 #181; L 安装介质到新的玻璃片. </li>
	<li> 用 P10 吸管, 将大脑从200和 #181 中转移; L 管到片, 旁边的安装介质下降。与组织一起, 转移一些 PBS, 以保持大脑在解决方案. </li>
	<li> 用 P10 吸管将大脑从 pbs 移到安装介质的下降, 试图限制 pbs 的数量。将试样在片上的安装介质上传送到成像间隔。使用足够的安装介质来覆盖试样. </li>
	<li> 从垫片的上部取下其它粘合剂衬垫, 并在试样顶部加一个玻璃片。用镊子的尖端, 施加一个柔和的压力在胶粘剂区域密封。立即将安装的组织图像或存储在-20 和 #176 的幻灯片; C 用于以后的分析. </li>
</ol> 7。图像获取 <ol> <li> 使用 40X (na = 1.2, 浸水) 目标或 63X (na = 1.4, 油浸) 目标共聚焦显微镜获取共焦荧光图像的堆栈 (像素大小 = 1024 x 1024 在 xy 平面上;两个共焦部分之间的距离 = 0.5 和 #181; m)。在每个通道中调整探测器增益和偏移, 使图像中不存在饱和像素和零像素值;这样可以避免信息丢失, 并确保使用探测器的全部动态范围. 
	注: 由于3D 成像耗时, 因此可以通过在不同位置并行扫描多个样本来自动进行测量。为了避免蒸发与水浸泡的目的, 使用特殊的油浸泡介质与折射率 n = 1.33. </li>
	<li> 使用任何标准图像分析软件处理最大密度投影、通道色调变化以及亮度和对比度的调整 (请参阅 材料表 ). </li>
</ol>

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M., Gaul, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glia+of+the+adult+Drosophila+nervous+system.">The glia of the adult Drosophila nervous system.</a> Glia. 65 (4), 606-638 (2017).</li></ol>

没有一个作者有竞争或相互冲突的利益。

该协议描述了一个简单和有效的方法来研究在高分辨率的兴趣组织内的不同细胞类型的形态学。采用 MCFO 技术, 多种不同表位标记的记者组合使用, 用于多色随机标记 (图 2)。与其他方法类似, 如 Brainbow/Flybow15,16,17, MCFO 通过标记共增加了标签的多样性, 从而使细胞边界的可视化成为细胞间相互作用的研究。?...

神经胶质是神经系统的神经细胞细胞, 长期以来被认为为神经元提供了一个静止的框架, 因此没有得到详细的研究。然而, 在人类中, 胶质细胞在 NS (约 90%) 中占绝大多数, 并分成几个不同的类别, 包括星状细胞、突、小胶质细胞和雪旺电池。在果蝇中, 胶质细胞在 NS 中占10% 左右。有趣的是, 它们的形态和功能与脊椎动物的1,2中发现的非常相似。它们的形态包括血脑屏障 (BBB) 形成上皮细胞、鞘层和星形星状胞。
果蝇中枢神经系统 (CNS) 包括以下主要结构: 包含神经元细胞体的皮层区域;neuropils 那海港突触连接;连接不同 neuropiles 的小的和大的轴突束;与中枢神经系统连接感觉器官和肌肉的外周神经 (图 1)。胶质细胞被发现与所有这些解剖结构相关: 皮质胶质细胞 (CG) 在皮层区, 星形胶质细胞和鞘胶质细胞 (如) 在 neuropile 区, 鞘胶质细胞也与中央轴突和周边神经 (地名), 最后, 两个片状胶质细胞, 膜胶质细胞 (PG) 和 subperineurial (), 其中一起形成一个连续的层覆盖整个 NS (图 2)。
以往的研究表明, 胶质细胞在 NS 的发展中起着重要的作用。它们通过对系统循环的胰岛素样肽进行反应来监测神经细胞的数量, 为神经元提供营养支持, 如星形胶质细胞-神经元乳酸梭, 并通过吞噬3,4来消除死亡神经元,5,6. 在成熟的 ns 中, 胶质细胞维持血脑屏障, 接受神经递质并维持离子稳态, 作为主要的免疫单元在 ns, 因为巨噬细胞不能破坏 bbb, 并调节突触活动以及动物行为6,7,8,9,10,11。
不同的胶质亚型是否具有特殊功能仍然是一个重要的开放性问题。然而, 对胶质细胞进行系统的全基因组分析, 特别是在成人身上, 由于缺乏适当的基因工具来进行操作而受到阻碍。在这里, 一个方法, 使细胞形状的有效和容易的表征研究复杂的细胞间的相互作用提出。这项技术已被用于表征不同的胶质亚型在成人果蝇脑中的形态学, 但是, 根据具体的 GAL4 驱动程序, 它可以适应研究神经元12,13, 任何类型的混合细胞, 原则上任何发展阶段的任何组织。

<table><tbody><tr><td>Water bath</td><td>Grant</td><td>GD100</td><td></td></tr><tr><td>PCR tubes</td><td>Sarstedt</td><td>72.737.002</td><td></td></tr><tr><td>Forceps</td><td>Dumont</td><td>11251-20</td><td></td></tr><tr><td>Dissecting dish 30 mm x 12 mmm</td><td>Electron Microscopy Sciences</td><td>70543-30</td><td>Glass dissection dish</td></tr><tr><td>Pyrex 3 Depression Glass Spot Plate</td><td>Corning</td><td>7223-34</td><td>Glass dissection plates</td></tr><tr><td>Sylgard Black</td><td>SYLGARD, Sigma-Aldrich</td><td>805998</td><td>home made with charcoal</td></tr><tr><td>ExpressFive S2 cell culture medium</td><td>Invitrogen</td><td>10486-025</td><td></td></tr><tr><td>20% PFA</td><td>Electron Microscopy Sciences</td><td>15713</td><td></td></tr><tr><td>Triton X-100</td><td>Roth</td><td>3051.3</td><td></td></tr><tr><td>Normal goat serum</td><td>Jackson Laboratories</td><td>005-000-121</td><td></td></tr><tr><td>Normal donkey serum</td><td>Jackson Laboratories</td><td>017-000-121</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A9647</td><td></td></tr><tr><td>Rabbit HA-tag</td><td>Cell Signaling</td><td>C29F4</td><td>Primary antibody, dilution 1:500</td></tr><tr><td>Rat FLAG-tag</td><td>Novus Biologicals</td><td>NBP1-06712</td><td>Primary antibody, dilution 1:100</td></tr><tr><td>Mouse V5-tag:DyLight 549</td><td>AdSerotec</td><td>0411</td><td>Conjugated antibody, dilution 1:200</td></tr><tr><td>anti-rabbit AlexaFluor 488</td><td>Invitrogen</td><td>A11034</td><td>Secondary antibody, dilution 1:250</td></tr><tr><td>anti-rat DyLight 647</td><td>Jackson Laboratories</td><td>712-605-153</td><td>Secondary antibody, dilution 1:100</td></tr><tr><td>Vecta Shield</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr><tr><td>SlowFate Gold</td><td>Invitrogen</td><td>S36937</td><td></td></tr><tr><td>Secure Seal Spacer</td><td>Grace Biolabs</td><td></td><td>Contact company for ordering</td></tr><tr><td>Microscope cover glass 22 X 60 mm</td><td>Marienfeld</td><td>101152</td><td></td></tr><tr><td>Microscope cover glass 22 x 22 mm</td><td>Roth</td><td>H874</td><td></td></tr><tr><td>Stereo Microscope, Leica MZ6</td><td>Leica</td><td></td><td></td></tr><tr><td>Confocal laser scanning microscope LSM710</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Immersol</td><td>Zeiss</td><td>518 F</td><td>Immersion oil for fluorescence-microscopy, halogen free</td></tr><tr><td>Immersol</td><td>Zeiss</td><td>W 2010</td><td>Immersion fluid for water-immersion objectives, halogen free</td></tr><tr><td>R56F03-GAL4 (EG)</td><td>Bloomington Stock Center</td><td>39157</td><td>GAL4 driver</td></tr><tr><td>R86E01-GAL4 (ALG)</td><td>Bloomington Stock Center</td><td>45914</td><td>GAL4 driver</td></tr><tr><td>hspFlpPestOpt; UAS-FRT-stop-FRT-myr-smGFP-HA, UAS-FRT-stop-FRT-myr-smGFP-FLAG, UAS-FRT-stop-FRT-myr-smGFP-V5</td><td>Bloomington Stock Center</td><td>64085</td><td>UAS reporter (https://bdscweb.webtest.iu.edu/stock/misc/mcfo.php)</td></tr><tr><td>Fiji (Image J)</td><td></td><td></td><td>Image analysis software</td></tr><tr><td>Multi Time Macro</td><td>Zeiss</td><td></td><td>Software for automated scanning</td></tr></tbody></table>

application of multicolor flpout technique to study high resolution single cell morphologies and cell interactions of glia in drosophila

细胞表现出不同的形态和复杂的解剖关系。细胞如何与邻居互动？单元格类型之间的交互是否不同, 甚至在给定类型中也有所不同？他们遵循什么样的空间规则？到目前为止, 由于缺乏用于高分辨率单细胞标记的工具, 这些基本问题的答案在体内受到了阻碍。在这里, 提供了一个详细的协议目标单细胞与多色 FlpOut (MCFO) 技术。这种方法依赖于三不同标记的记者 (HA, 旗帜和 V5) 在无人控制, 保持沉默的转录终结者两侧的两个首次登记税网站 (首次登记-停止-首次登记税)。热休克脉冲诱发热休克诱导的 Flp 重组的表达, 它在单个单元格中随机删除了首次登记停用的卡带: 表达仅发生在同时表达 GAL4 驱动程序的单元格中。这将导致一个给定单元格类型的不同颜色的单元格数组, 它允许在高分辨率下可视化单个细胞形态。作为一个例子, MCFO 技术可以结合特定的胶质 GAL4 驱动, 以可视化的形态, 不同的胶质亚型在成年果蝇脑。

本节说明了在成人果蝇脑中使用 MCFO 技术可以获得的结果的示例。 图 3显示了该方法的示意图。三不同的膜标记的记者 (myr-smGFP-HA, myr-smGFP-旗和 myr-smGFP-V5) 在无人控制系统下, 由两个首次登记税网站 (首次登记-停止-首次登记税) 两侧的转录终结者保持沉默。热休克脉冲诱导 Flp 重组的表达, 它在单个单元格中随机删除了首次登记停用...

Watch this Scientific Journal Video about Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila at JoVE.com

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

细胞表现出不同的形态, 并与它们的邻居建立了各种相互作用。该协议描述了如何揭示单细胞的形态学, 并利用已建立的 Gal4/UAS 表达系统来研究细胞间的相互作用。

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 ...

application-multicolor-flpout-technique-to-study-high-resolution

Universidad San Sebastian

Research

JoVE Journal

Behavior

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

8.1K 次观看.  Ludwig-Maximilians-University Munich. 细胞表现出不同的形态, 并与它们的邻居建立了各种相互作用。该协议描述了如何揭示单细胞的形态学, 并利用已建立的 Gal4/UAS 表达系统来研究细胞间的相互作用。

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

Single Drosophila Ommatidium Dissection and Imaging

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative diseases because of its powerful genetics1. The fly eye in particular has been the organ of choice for neurodegeneration research, being the most accessible and life-dispensable part of the Drosophila nervous system. However the major caveat of intact eyes is the difficulty, because of the intense autofluorescence of the pigment, in imaging intracellular events, such as autophagy dynamics2, which are paramount to understanding of neurodegeneration.
We have recently used the dissection and culture of single ommatidia3 that has been essential for our understanding of autophagic dysfunctions in a fly model of Dentatorubro-Pallidoluysian Atrophy (DRPLA)3, 4.
We now report a comprehensive description of this technique (Fig. 1), adapted from electrophysiological studies5, which is likely to expand dramatically the possibility of fly models for neurodegeneration. This method can be adapted to image live subcellular events and to monitor effective drug administration onto photoreceptor cells (Fig. 2). If used in combination with mosaic techniques6-8, the responses of genetically different cells can be assayed in parallel (Fig. 2).

Single Drosophila Ommatidium Dissection and Imaging

The limiting factor in the use of the adult Drosophila eye to study neurodegeneration and cell biology is the difficult imaging of intracellular processes. We describe the dissection of single ommatidia to generate a bona-fide primary neuronal cell culture, which can be subject to drug treatment and advanced imaging.

单果蝇 Ommatidium解剖和影像

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative ...

科研

神经科学

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

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

可视化的Live果蝇胶质细胞神经​​肌肉交界处，与荧光染料

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

生物学

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

住在果蝇眼睛成虫光盘胶质细胞迁移成像

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

标记的单细胞在中枢神经系统<em&gt;果蝇</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila larvae, and fish, which all engage in undulatory movements (like crawling or swimming) as their primary mode of locomotion. However, a more sophisticated understanding of the individual motor neuron (MN) specification&#8212;at least in terms of informing disease therapies&#8212;demands an equally tractable system that better models the complex appendage-based locomotion schemes of vertebrates. The adult Drosophila locomotor system in charge of walking meets all of these criteria with ease, since in this model it is possible to study the specification of a small number of easily distinguished leg MNs (approximately 50 MNs per leg) both using a vast array of powerful genetic tools, and in the physiological context of an appendage-based locomotion scheme. Here we describe a protocol to visualize the leg muscle innervation in an adult fly.

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

Here we describe a protocol to visualize the axonal targeting with a florescent protein in adult legs of Drosophila by fixation, mounting, imaging, and post-imaging steps.

通过成人角质层可视化果蝇腿运动神经元轴突

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila ...

Applications of Immobilization of Drosophila Tissues with Fibrin Clots for Live Imaging

Drosophila is an important model system to study a vast range of biological questions. Various organs and tissues from different developmental stages of the fly such as imaginal discs, the larval brain or egg chambers of adult females or the adult intestine can be extracted and kept in culture for imaging with time-lapse microscopy, providing valuable insights into cell and developmental biology. Here, we describe in detail our current protocol for the dissection of Drosophila larval brains, and then present our current approach for immobilizing and orienting larval brains and other tissues on a glass coverslip using Fibrin clots. This immobilization method only requires the addition of Fibrinogen and Thrombin to the culture medium. It is suitable for high-resolution time lapse imaging on inverted microscopes of multiple samples in the same culture dish, minimizes the lateral drifting frequently caused by movements of the microscope stage in multi-point visiting microscopy and allows for the addition and removal of reagents during the course of imaging. We also present custom-made macros that we routinely use to correct for drifting and to extract and process specific quantitative information from time-lapse analysis.

Applications of Immobilization of Drosophila Tissues with Fibrin Clots for Live Imaging

This protocol describes applications of sample immobilization using Fibrin clots, limiting drifting, and allowing the addition and washout of reagents during live imaging. Samples are transferred to a drop of Fibrinogen containing culture medium on the surface of a coverslip, after which polymerization is induced by adding thrombin.

使用纤维素凝块固定 的果蝇 组织用于实时成像

Drosophila is an important model system to study a vast range of biological questions. Various organs and tissues from different developmental stages of ...

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of embryogenesis, these organelles undergo dramatic movements powered by actin-based cytoplasmic streaming and motor-driven trafficking along microtubules. The development of a multitude of small, organelle-specific fluorescent probes (FPs) makes it possible to visualize a wide range of different lipid-containing structures in any genotype, allowing live imaging without requiring a genetically encoded fluorophore. This protocol shows how to inject vital dyes and molecular probes into Drosophila embryos to monitor the trafficking of specific organelles by live imaging. This approach is demonstrated by labeling lipid droplets (LDs) and following their bulk movement by particle image velocimetry (PIV). This protocol provides a strategy amenable to the study of other organelles, including lysosomes, mitochondria, yolk vesicles, and the ER, and for tracking the motion of individual LDs along microtubules. Using commercially available dyes brings the benefits of separation into the violet/blue and far-red regions of the spectrum. By multiplex co-labeling of organelles and/or cytoskeletal elements via microinjection, all the genetic resources in Drosophila are available for trafficking studies without the need to introduce fluorescently tagged proteins. Unlike genetically encoded fluorophores, which have low quantum yields and bleach easily, many of the available dyes allow for rapid and simultaneous capture of several channels with high photon yields.

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

In the early Drosophila embryo, many organelles are motile. In principle, they can be imaged live via specific fluorescent probes, but the eggshell prevents direct application to the embryo. This protocol describes how to introduce such probes via microinjection, and then analyze bulk organelle motion via particle image velocimetry.

可视化 果蝇 胚胎中含脂质细胞器的细胞骨架依赖性运输

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of ...

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

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. The molecular clock within each pacemaker neuron generates circadian rhythms in gene expression, which underlie the rhythmic neuronal functions essential to the operation of the circuit. Investigation of the properties of the individual molecular oscillators in different subclasses of pacemaker neurons and their interaction with neuronal signaling yields a better understanding of the circadian pacemaker circuit. Here, we present a time-lapse fluorescent microscopy approach developed to monitor the molecular clockwork in clock neurons of cultured Drosophila larval brain. This method allows the multi-day recording of the rhythms of genetically encoded fluorescent circadian reporters at single-cell resolution. This setup can be combined with pharmacological manipulations to closely analyze real-time response of the molecular clock to various compounds. Beyond circadian rhythms, this multipurpose method in combination with powerful Drosophila genetic techniques offers the possibility to study diverse neuronal or molecular processes in live brain tissue.

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

The goal of this protocol is to establish ex vivo Drosophila larval brain culture optimized to monitor circadian molecular rhythms with long-term fluorescence time-lapse imaging. The application of this method to pharmacological assays is also discussed.

单细胞分辨荧光活成像在幼虫脑培养中的果蝇昼夜节律时钟

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

Multiplex Detection of Gene Expression in the Intact Drosophila Brain Using Expansion-Assisted Iterative Fluorescence In Situ Hybridization

Understanding gene expression is essential for deciphering cellular functions. However, methods for analyzing the expression of numerous genes in situ within a given tissue remain limited. The EASI-FISH protocol described here has been adapted to detect the expression of dozens of genes in the intact adult Drosophila central nervous system (CNS) using commercially available reagents. This protocol includes a new gel formulation that enhances gel robustness, enabling multiple rounds of hybridization and allowing the embedding of multiple brains per gel. This improvement increases throughput, facilitates optimal comparison of experimental conditions, and reduces reagent costs. Additionally, by employing the GAL4-UAS system for co-detection of green fluorescent protein (GFP), gene expression can be visualized in specific neuronal or glial cell types. Notably, the high resolution achieved through expansion microscopy, combined with the sensitivity of the method, allows for the detection of single RNA transcripts. This approach effectively integrates high image quality with high throughput, making it a powerful tool for studying gene expression throughout the intact fly brain.

Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) is a robust technique that combines expansion microscopy with fluorescence in situ hybridization (FISH) to detect the expression of multiple genes in thick tissue. This protocol outlines recent advancements in the method and its adaptation for labeling the intact Drosophila central nervous system..

Understanding gene expression is essential for deciphering cellular functions. However, methods for analyzing the expression of numerous genes in situ ...

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

摘要

探索更多视频

此视频中的章节

单果蝇 Ommatidium解剖和影像

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

可视化的Live果蝇胶质细胞神经肌肉交界处，与荧光染料

住在果蝇眼睛成虫光盘胶质细胞迁移成像

标记的单细胞在中枢神经系统

通过成人角质层可视化果蝇腿运动神经元轴突

使用纤维素凝块固定的果蝇组织用于实时成像

可视化果蝇胚胎中含脂质细胞器的细胞骨架依赖性运输

单细胞分辨荧光活成像在幼虫脑培养中的果蝇昼夜节律时钟

Multiplex Detection of Gene Expression in the Intact Drosophila Brain Using Expansion-Assisted Iterative Fluorescence In Situ Hybridization

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

摘要

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此视频中的章节

单果蝇 Ommatidium解剖和影像

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

可视化的Live果蝇胶质细胞神经​​肌肉交界处，与荧光染料

住在果蝇眼睛成虫光盘胶质细胞迁移成像

标记的单细胞在中枢神经系统

通过成人角质层可视化果蝇腿运动神经元轴突

使用纤维素凝块固定 的果蝇 组织用于实时成像

可视化 果蝇 胚胎中含脂质细胞器的细胞骨架依赖性运输

单细胞分辨荧光活成像在幼虫脑培养中的果蝇昼夜节律时钟

Multiplex Detection of Gene Expression in the Intact Drosophila Brain Using Expansion-Assisted Iterative Fluorescence In Situ Hybridization

可视化的Live果蝇胶质细胞神经肌肉交界处，与荧光染料

使用纤维素凝块固定的果蝇组织用于实时成像

可视化果蝇胚胎中含脂质细胞器的细胞骨架依赖性运输