Name: preparing developing peripheral olfactory tissue for molecular and immunohistochemical analysis in drosophila
Uploaded: 2018-06-13T14:30:00.000+00:00
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Description: Watch this Scientific Journal Video about Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila at JoVE.com

这项研究得到了国家科学基金会对 Pelin c 沃尔坎·武拉尔 (DEB-1457690) 的资助。

以下议定书符合杜克大学研究伦理学委员会制定的道德准则。
1.果蝇蛹的组织制备及发育分期
<ol><li>首先, 确定解剖需要多少苍蝇。对于 RT PCR 和 RNAseq, 收集 100-200 触角光盘和触角的个人, 这是必要的 prepupal, 8 h apf, 40 h 有源电力, 和成人阶段。免疫组化, 解剖 10-20 个人。</li>
	<li>清洁所有材料, 包括解剖垫和镊子, 使用70% 乙醇的湿巾。如果被解剖的材料用于 RNA 提取, 清洁所有与 RNase 中和剂并且使用无核酸酶或二乙基焦碳酸 (DEPC) 处理的水的所有解答。 注: 本协议使用硅胶夹层垫代替玻璃进行解剖。硅胶垫是友好的超细钳和促进快速和高质量的解剖。</li>
	<li>在 0 h 型 APF/prepupal 阶段采集蛹。<ol>
<li>为了确保最精确的分期, 监测幼虫在30分钟至1小时间隔。 注意: 在适度拥挤的瓶子里游荡的第三龄幼虫可能会在几个小时内蛹。</li>
		<li>一旦幼虫变得不动, 被一个非常轻 (几乎是白色) 的彩色蛹壳所包围, 将它们转移到一个小的2在培养皿中, 带有湿润的滤纸。</li>
		<li>继续 1-2 小时, 偶尔监视、识别和转移预蛹。 注: 另外, 清除一瓶所有蛹, 并允许幼虫发育2小时。所有2小时后收集的蛹将在 0-2 h APF 范围内。</li>
	</ol>
</li>
	<li>立即移动到步骤 3.1, 以解剖 prepupal 触角盘为 0 h APF 阶段。如果预蛹需要老化, 把它们收集到盘子里, 盖上盘子, 在边缘周围放置一层石蜡膜, 以抑制干燥, 并将培养皿放在25摄氏度。 注: 蛹现在可以老化到羽化。</li>
	<li>对于在2小时范围内收集的蛹, 年龄预蛹为2小时小于所需年龄, 以确保蛹不过剩。</li>
	<li>使用超细钳 (55), 因为组织是非常小和脆弱的。</li>
</ol>2. 其他果蝇品种的组织制备和蛹的发育分期
<ol><li>为了确定其他果蝇物种蛹发育的长度, 请将样品放在最佳温度下, 记录预蛹观察到的日期, 将它们分类为新鲜的小瓶, 并记录从预蛹到成年的天数。</li>
	<li>记录非黑腹和蛹的发育时间比例. 将发育时间的比例乘以用于腹腹的小时数, 以获得非黑腹动物的年龄.</li>
</ol>3. 蛹触角瓣的解剖
注: 所有老化时间和解剖协议都描述为果蝇.就所有其他物种而言, 如上文所述, 需要根据物种特定的发展时间点修改老化协议。
<ol><li>对于蛹触角椎间盘解剖, 收集 50-100 0-2 小时或 6-8 h 老蛹使用铲, 并把它们放在解剖垫上。</li>
	<li>吸管150µL 的 RNA 隔离溶液成一个 RNase 自由1.5 毫升离心管使用200µL 吸管, 并把管放在冰上。</li>
	<li>解剖蛹在 150-200 µL 1x PBS (磷酸盐缓冲盐水, 核酸酶)。在解剖垫上放置多滴 PBS (每滴 150-200 µL)。</li>
	<li>在解剖垫上, 放置 0-2 小时或 6-8 小时的蛹 (一个蛹每滴) 被解剖在 1x PBS 水滴之一。用钳子一只手来稳定身体。</li>
	<li>对于 0-2 小时的蛹, 使用镊子, 另一方面, 抓住最前面的部分 (口钩) 的预蛹, 并拉出它暴露的大脑和形象光盘附着在前蛹的情况下。</li>
	<li>对于 8 h 型有源性蛹, 首先从蛹的最前节轻轻剥下蛹的外壳, 将头部暴露在身体周围的膜袋内。 注意: 身体, 在这个阶段, 看起来像一个退化的幼虫。</li>
	<li>把头移到颈部关节上。 注意: 这确保了触角光盘不会丢失, 在这个阶段, 主要是连接到大脑的视神经裂片。</li>
	<li>使用镊子或20µL 吸管将组织与大脑和形象盘转移到另一 1x PBS 液滴上。</li>
	<li>识别与大脑在视神经裂片上连接的眼触角盘。使用镊子, 从大脑和蛹的情况下拔下眼触角盘。使用镊子或20µL 吸管将其转移到新的 1x PBS 液滴上。 注: prepupal 眼触角盘为扁平组织。然而, 在8小时的蛹, 眼球被旋转拔罐的触角盘, 它位于中央大脑前。</li>
	<li>使用镊子, 切除眼睛和触角之间的连接部位的组织。使用20µL 吸管将解剖组织轻轻地转移到 RNA 隔离溶液试剂中。尽量减少吹打转移的液体数量。</li>
	<li>重复, 直到所有的蛹被解剖。</li>
</ol>4. 蛹和成人触角的解剖
注: 由 40 h 型 APF, 大多数变形是完整的, 成人结构变得明显, 但仍然是白色和无可名状的。在这个发展的时间点, 成年天线已经采取了它的最终形状, 但仍然是透明的, 大多数的嗅觉受体, 除了少数, 尚未开始表达。
<ol><li>对于蛹触角解剖, 收集 50-100 预蛹描述在步骤1和年龄他们为40小时在25°c。</li>
	<li>吸管150µL 的 RNA 隔离溶液成一个 RNase 自由1.5 毫升离心管使用200µL 吸管, 并把管放在冰上。</li>
	<li>在解剖垫上, 将蛹解剖成 1x PBS 液滴之一。用钳子一只手来稳定身体。另一方面, 使用镊子, 轻轻剥去从蛹的最前一季度蛹的情况下, 暴露头部内的膜袋, 周围的身体。</li>
	<li>使用镊子, 小心地切断从其余的蛹身体的头部, 通过持有一组钳和撕裂头在颈部关节与另一套镊子。用镊子轻轻将头部转到另一 1x PBS 液滴上。用吸管向上和向下清理头部 (大脑等) 的内容, 以获得用于环绕显影果蝇头部的透明膜。 注: 在 40-50 h 型 APF 中, 触角连接到膜的最前面部分。当头部的内容与吹打清除时, 它们将变得明显。</li>
	<li>使用镊子, 从膜上拔下蛹触角。将天线的 2 和段从 3路拔下, 用镊子在节段关节上切片, 因为只有 3路段将嗅到受体神经元。</li>
	<li>使用20µL 吸管将解剖组织轻轻地转移到 RNA 隔离溶液试剂中。尽量减少吹打转移的液体数量。重复, 直到所有的蛹被解剖。</li>
	<li>成人触角解剖, 收集大约50成人和年龄他们在羽化后一周。 注: 嗅觉受体基因和其他基因的表达在羽化后一周达到峰值。成年人的年龄为一周, 以确保生物学相关的基因与低丰度转录高于检测阈值。</li>
	<li>对于 RNA 提取, 解剖苍蝇, 而他们仍然活在 CO2垫。用 RNase 抑制剂治疗 CO2垫。为共焦成像, 解剖在解剖垫上的苍蝇在 1x pbs 或 pbs + 0.2% 海卫一。</li>
	<li>首先, 把成年人睡在 CO2站垫的解剖范围内。在 CO2站垫上, 一方面使用镊子来稳定身体, 另一方面用镊子轻轻拉/捏出第三个触角段。</li>
	<li>将触角转移到 RNA 隔离溶液中, 用镊子将触角直接放进液体中。重复, 直到所有成年人被解剖。 注意: 确保天线被淹没在 RNA 隔离溶液中。天线可能会卡在管子的侧面。这将导致 rna 的退化增加, 降低 rna 的质量和数量。</li>
	<li>直接进行 RNA 提取或放置在-80 摄氏度的管长期贮存。</li>
</ol>5. RNA 分离从被解剖的组织
<ol><li>从-80 摄氏度的储藏中取出所有样品, 然后在冰上解冻。</li>
	<li>简要地旋转 (5-6 s, ~ 6000 x g) 每个样品收集在管子的底部的材料。</li>
	<li>在冰上使用蒸压塑料杵和一台电动机在十年代研磨每个样品。</li>
	<li>重复步骤5.2 和 5.3 4。</li>
	<li>使用商业上可用的套件执行 RNA 提取, 并按照制造商的协议获得最佳产量。</li>
	<li>使用商业上可用的试剂和特定的引物对感兴趣的基因进行 qRT PCR, 并遵循制造商的协议。<ol>
<li>使用以下条件进行所有 PCR 反应:95 °c 变性为10分钟其次40个周期95°c 为十五年代, 55 °c 三十年代和60°c 为三十年代。 注: 在表 1中列出了浸出和休斯敦基因的底漆对, 并列出了嗅觉受体基因的引物 。8。</li>
	</ol>
</li>
</ol>6. 蛹触角盘和触角的免疫组化治疗
注: 从 10-20 人的批次中固定组织, 以确保组织在同一时间内修复。将样品停留在 PBT (PBS 和洗涤剂) 的时间减到最小, 然后将其转移到固定件, 以最大限度地提高组织的完整性。
<ol><li>在200µL 0.2% PBT 的 200-µL pcr 管中收集解剖触角盘或蛹天线, 以保持组织完整性, 并避免样品粘附在 200-µL pcr 管壁上, 从而导致不完整的固定。 注意: 蛹天线和触角光盘可能很难看到, 因为它们是相当半透明的。最好使用解剖范围的所有洗涤步骤, 以防止任何损失的组织。另外, 96 井 Terasaki 板可用于固定和染色, 以最佳跟踪组织。</li>
	<li>在室温下, 将大约200µL 4% 粉煤灰 (多聚甲醛) 的蛹中的解剖触角盘和触角标本固定在30分钟内。对于此和后续步骤, 请将管道与 nutator 上的示例保持在一起, 以便正确地混合解决方案中的示例。步骤6.7 的进度。</li>
	<li>对于成人天线, 首先解剖整个头部, 并在室温下将其固定在大约200µL 4% 粉煤灰 (多聚甲醛) 中1小时。</li>
	<li>洗涤3x 与200µL 0.2% PBT 为10分钟每。在孵化过程中, 将样品保存在 nutator 上。</li>
	<li>回到解剖显微镜, 以更好的解剖第二触角段从固定头。使用镊子稳定头部, 轻轻拉/捏出第三触角段从第二。</li>
	<li>将解剖后的第三触角段转移到大约200µL 4% 粉煤灰 (多聚甲醛) 中, 并在室温下将其固定30分钟。</li>
	<li>洗涤3x 与200µL 0.2% PBT 为10分钟每。在孵化过程中, 将样品保存在 nutator 上。</li>
	<li>将样品保存在4摄氏度或直接与整个芒特免疫组化。 注意: 当使用抗体时, 染色效果可能会改变。这种方法应该适合于不太丰富的蛋白质标签或较弱的抗体。然而, 有时, 它可能需要优化的协议 (无论是身份或数量的固定器或的时间)。</li>
</ol>

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作者没有什么可透露的。

该协议是一个有用的资源, 实验室有兴趣确定的基因和转录程序在开发果蝇嗅觉组织, 以及对这些项目的比较分析果蝇物种。如果需要从不同发育阶段的系统级转录剖面作为未来研究的入门资料, 则特别有用。这里描述的协议演示了如何分期和隔离开发果蝇的嗅觉组织, 从从前阵列到末端分化的四个关键阶段从嗅觉系统开发中获取样本, 用于分子和免疫组织学研究。解剖果蝇</...

果蝇嗅觉系统是发展神经生物学、神经系统神经系统学、神经生理学、行为研究和行为进化1、2、3的一种广泛应用的系统, 4。果蝇嗅觉组织家的嗅觉受体神经元 (ORNs), 检测挥发性化学线索除了水力和热感官神经元1,5,6。本手稿的总体目标是演示如何在果蝇物种中进行蛹和成人嗅觉组织的分期和解剖。这种技术的基本原理是从不同的蛹阶段生成样本, 用于对外围嗅觉系统进行分子和发展分析, 如 RNAseq 和定量 rt-pcr, 除了体内组织标记技术.这项技术对于识别发育中的成人嗅觉系统中非腹果蝇物种的转录剖面的动力学特别有用, 因为它没有所有的细胞类型特定的转基因试剂。标签和分析。在这篇手稿中, 我们演示如何解剖触角光盘和触角从蛹和成年果蝇物种。首先, 我们展示了如何识别果蝇0 h 的蛹壳形成 (APF, 白蛹或预蛹) 的发展分期和物种特定的衰老。接下来, 我们展示了如何解剖触角盘和第三触角段;具体来说, 如何将它们从眼盘和第二触角段中分离出来, 以 RNAseq 或 RT PCRs 所需的组织纯度。本协议中描述的D.触角的阶段大致对应于预阵列 (预蛹), 从触角盘 (8 h apf) 中选取前体, ORNs (40 h apf) 的终端分化开始, 以及成人天线。最后, 给出了用该方法分离成人触角的定量 rt-pcr 的例子, 并对体内免疫组化进行了研究。该方法可用于生成 RNA/DNA 分析样本和免疫组化技术, 用于开发不同果蝇物种的外周嗅觉组织。

<table><tbody><tr><td>10X Phosphate-buffered Saline</td><td>Gibco</td><td>70011-044</td><td>Dilute to 1X in RNase free water</td></tr><tr><td>CO&lt;sub&gt;2 &lt;/sub&gt;tank</td><td>Air Gas</td><td>CD R200</td><td></td></tr><tr><td>Two sharp forceps (Dumont #55)</td><td>Fine Science Tools</td><td>11255-20</td><td></td></tr><tr><td>Trizol</td><td>Invitrogen</td><td>15596-026</td><td>RNA isolation solutions</td></tr><tr><td>Dissecting Microscope</td><td></td><td></td><td></td></tr><tr><td>Small bucket with ice</td><td></td><td></td><td></td></tr><tr><td>P20 and P200 micropipettes and tips</td><td></td><td></td><td></td></tr><tr><td>1.7 mL Microfuge tubes</td><td></td><td></td><td>Purchase nuclease free for best results</td></tr><tr><td>0.2 mL PCR tubes</td><td></td><td></td><td></td></tr><tr><td>Paraformaldehyde powder</td><td>Polysciences</td><td>380</td><td></td></tr><tr><td>10% Triton X-100</td><td>Teknova</td><td>T1105</td><td>Dilute to 0.2% v/v in 1X PBS</td></tr><tr><td>2&quot; petri dishes</td><td></td><td></td><td></td></tr><tr><td>55mm filter paper</td><td>Whatman</td><td>1001-055</td><td>Wet with water and place in a petri dish to keep pupae moist</td></tr><tr><td>25 C incubator</td><td></td><td></td><td></td></tr><tr><td>deionized H2O</td><td></td><td></td><td>Purchase nuclease free or treat with DEPC</td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning</td><td></td><td>to be used for making dissection pads from Terizaki plate covers.</td></tr><tr><td>MicroWell Mini Trays with lids</td><td>Nunc</td><td>438733</td><td>Lids can be used to make silicone dissection pads</td></tr><tr><td>Rabbit anti-GFP antibody</td><td>MBL international corpor</td><td>PM005</td><td>primary antibody</td></tr><tr><td>Mouse anti RAT CD2</td><td>AbD seroTec</td><td>MCA154RHDL</td><td>primary antibody</td></tr><tr><td>ADL195 (anti lamin, mouse)</td><td>Dev studies hydoma ban</td><td>ADL195-s</td><td>primary antibody</td></tr><tr><td>Alexa Fluor&amp;reg; 488 goat anti-rabbit IgG (H+L)</td><td>invitrogen</td><td>A11008</td><td>Secondary antibody</td></tr><tr><td>Cy3 Goat Anti-Mouse IgG</td><td>Jackson ImmunoResearch</td><td>115-166-003</td><td>Secondary antibody</td></tr><tr><td>Triton X-100, Protein Grade Detergent, 10% Solution, Sterile-Filtered</td><td>CALBIOCHEM</td><td>648463</td><td>detergent</td></tr><tr><td>Lab Rotator</td><td>Thermo scientific</td><td></td><td>Rotator</td></tr><tr><td>Nuclease Free Water</td><td>Growcells.com</td><td>NUPW-0125</td><td>Water</td></tr><tr><td>Light-Duty Tissue Wipers</td><td>VWR</td><td>82003-822</td><td>For cleaning dissection supplies</td></tr><tr><td>Superscript II</td><td>invitrogen</td><td>18064014</td><td>Reverse Transcriptase&amp;nbsp;</td></tr><tr><td>QIAshredder (50)</td><td>RNA extraction</td><td>QIAGEN</td><td>79654</td></tr><tr><td>Oligo(dT)12-18 Primer</td><td>RT</td><td>life technologies</td><td>18418012</td></tr><tr><td>RNeasy MinElute Cleanup Kit (50)</td><td>RNA extraction</td><td>QIAGEN</td><td>74204</td></tr><tr><td>FASTSTART UNIV SG MASTER (5 ML)(500 RXN)</td><td>qPCR</td><td>Roche</td><td>04913850001</td></tr><tr><td>FASTSTART ESSENTIAL GREEN DNA MASTER&amp;nbsp;</td><td>qPCR</td><td>Roche</td><td>06402712001</td></tr><tr><td>LIGHTCYCLER 480 - MULTIWELL PLATE 96</td><td>qPCR</td><td>Roche</td><td>04729692001</td></tr><tr><td>NEBNext High-Fidelity 2X PCR Master Mix</td><td>qPCR</td><td>NEB</td><td>M0541S</td></tr></tbody></table>

preparing developing peripheral olfactory tissue for molecular and immunohistochemical analysis in drosophila

果蝇嗅觉系统是发展神经生物学、神经系统神经系统学、神经生理学、行为学和行为进化等广泛应用的系统。果蝇嗅觉组织家的嗅觉受体神经元 (ORNs), 检测挥发性化学线索, 除了水力和热感官神经元。在本议定书中, 我们描述了发展成人果蝇周围嗅觉组织的解剖。我们首先描述如何分期和年龄果蝇幼虫, 其次是解剖的触角盘从早期蛹阶段, 其次是解剖的触角从中蛹阶段和成人。我们还展示了在分子技术中可以利用制剂的方法, 如 RNA 提取 qRT PCR、RNAseq 或免疫组化。这些方法也可以应用于其他果蝇物种后, 物种特定的蛹开发时间确定, 并为适当的老化计算各个阶段。

经解剖开发的嗅觉组织可用于 RNA 提取, 其次是 rt-pcr 评估基因表达, 或通过免疫组化协议, 以确定其表达模式, 以及基因的亚细胞定位兴趣。在本节中, 我们从任一议定书中提出代表的结果。图 1是典型的定量 rt-pcr, 显示了野生型成人触角中细胞表面受体和嗅觉受体基因的转录。图 2显示了染色 (1:1000) 和抗 lamin 抗体 (1:100) (<strong cl...

Watch this Scientific Journal Video about Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila at JoVE.com

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

在这里, 我们提出了一个阶段和解剖发展的嗅觉组织从果蝇物种的协议。解剖后的组织可用于分子分析, 如定量 rt-pcr (反向转录-聚合酶链反应) 或 RNA 测序 (RNAseq), 以及体内分析, 如免疫组化或原位杂交。

果蝇分子和免疫组化分析制备周围嗅组织的研制

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and ...

preparing-developing-peripheral-olfactory-tissue-for-molecular

Research

JoVE Journal

Developmental Biology

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

5.7K 次观看.  Duke University. 在这里, 我们提出了一个阶段和解剖发展的嗅觉组织从果蝇物种的协议。解剖后的组织可用于分子分析, 如定量 rt-pcr (反向转录-聚合酶链反应) 或 RNA 测序 (RNAseq), 以及体内分析, 如免疫组化或原位杂交。

视频: Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

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

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response to&#160;growth and reproduction. In insects, the larval prothoracic gland (PG) and the adult female ovary play essential roles in biosynthesizing the principal steroid hormones called ecdysteroids. These ecdysteroidogenic organs are innervated from the nervous system, through&#160;which the timing of biosynthesis is affected by environmental cues. Here we describe a protocol for visualizing ecdysteroidogenic organs and their interactive organs in larvae and adults of the fruit fly Drosophila melanogaster, which provides a suitable model system for studying steroid hormone biosynthesis and its regulatory mechanism. Skillful dissection allows us to maintain the positions of ecdysteroidogenic organs and their interactive organs including the brain, the ventral nerve cord, and other tissues. Immunostaining with antibodies against ecdysteroidogenic enzymes, along with transgenic fluorescence proteins driven by tissue-specific promoters, are available to label ecdysteroidogenic cells. Moreover, the innervations of the ecdysteroidogenic organs can also be labeled by specific antibodies or a collection of GAL4 drivers in various types of neurons. Therefore, the ecdysteroidogenic organs and their neuronal connections can be&#160;visualized simultaneously&#160;by immunostaining and transgenic techniques. Finally, we describe how to visualize germline stem cells, whose proliferation and maintenance are controlled by ecdysteroids. This method contributes to comprehensive understanding of steroid hormone biosynthesis and its neuronal regulatory mechanism.

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

We describe a protocol for dissection, fixation, and immunostaining of steroidogenic organs in Drosophila larvae and adult females to study steroid hormone biosynthesis and its regulatory mechanism. In addition to steroidogenic organs, we visualize the innervation of steroidogenic organs as well as steroidogenic target cells such as germline stem cells.

协议的可视化类固醇机关及其互动器官在果蝇免疫染色<em&gt;果蝇</em

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response ...

发育生物学

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval body wall contains several cell types that are models to study cell and tissue morphogenesis. For example tracheae are used to examine tube morphogenesis1, and the dendritic arborization (DA) sensory neurons of the Drosophila larva have become a primary system for the elucidation of general and neuron-class-specific mechanisms of dendritic differentiation2-5 and degeneration6.
The shape of dendrite branches can vary significantly between neuron classes, and even among different branches of a single neuron7,8. Genetic studies in DA neurons suggest that differential cytoskeletal organization can underlie morphological differences in dendritic branch shape4,9-11. We provide a robust immunological labeling method to assay in vivo microtubule organization in DA sensory neuron dendrite arbor (Figures 1, 2, Movie 1). This protocol illustrates the dissection and immunostaining of first instar larva, a stage when active sensory neuron dendrite outgrowth and branching organization is occurring 12,13.
In addition to staining sensory neurons, this method achieves robust labeling of microtubule organization in muscles (Movies 2, 3), trachea (Figure 3, Movie 3), and other body wall tissues. It is valuable for investigators wishing to analyze microtubule organization in situ in the body wall when investigating mechanisms that control tissue and cell shape.

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

To understand how complex cell shapes, such as neuronal dendrites, are achieved during development, it is important to be able to accurately assay microtubule organization. Here we describe a robust immunohistological labeling method to examine microtubule organization of dendritic arborization neuron sensory dendrites, trachea, muscle, and other Drosophila larva body wall tissues.

免疫组化标记在感觉神经元树突的微管，气管，并在肌肉果蝇幼虫体壁

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval ...

神经科学

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction.&#160;

嗜酸性粒植物视网膜免疫组织化学、西方分析和 Rna 分离的解剖

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface ('rough eye' phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.1). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes.
The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g. 2). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells ('hexagon' around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six "outer" (R1-6) and two "inner" photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior and dorsoventral axes of the eye (Fig. 3A). In particular, the ommatidia of the dorsal and ventral (black and red arrows, respectively) halves of the eye are mirror images of each other and correspond to two chiral forms established during planar cell polarity signaling (for review see e.g. 3).
The method to generate semi-thin eye sections (such as those presented in Fig. 3) described here is slightly modified from the one originally described by Tomlinson and Ready4. It allows the morphological analysis of all cells except for the transparent cone cells. In addition, the pigment of R-cells (blue arrowheads in Fig. 2 and 3) can be used as a cell-autonomous marker for the genotype of a R-cell, thus genetic requirements of genes in a subset of R-cells can readily be determined5,6.

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

A standard approach to prepare adult Drosophila eyes for semi-thin sectioning and light microscopic analysis is presented here. The protocol can be used for gross morphological analysis of eye defects, or with the indicated adjustments can be used to determine genetic requirements of genes in specific cell types of the eye (e.g. clonal analysis of photoreceptors) or for electron microscopic analysis.

成人的制备果蝇</em薄切片和显微镜分析>的眼睛

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a ...

生物学

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, oviposition, and mating. Myriad chemicals of varying volatilities have been identified as natural odorants that activate insect Olfactory Receptor Neurons (ORNs). However, studying the olfactory responses to low-volatility odorants has been hampered by an inability to effectively present such stimuli using conventional odor-delivery methods. Here, we describe a procedure that permits the effective presentation of low-volatility odorants for in vivo Single-Sensillum Recording (SSR). By minimizing the distance between the odor source and the target tissue, this method allows for the application of biologically salient but hitherto inaccessible odorants, including palmitoleic acid, a stimulatory pheromone with a demonstrated effect on ORNs involved in courtship and mating behavior1. Our procedure thus affords a new avenue to assay a host of low-volatility odorants for the study of insect olfaction and pheromone communication.

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

The overall goal of this protocol is to demonstrate how to present odorants of low volatility for single-sensillum recording from Drosophila olfactory receptor neurons that respond to long-chain cuticular pheromones.

电生理记录<em&gt;果蝇</em&gt; Trichoid Sensilla响应低挥发性气味

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, ...

<meta charset="utf8"></head><body>嗅觉体验诱导的果 蝇 幼年脑突触重塑

Experience-Dependent Remodeling of Juvenile Brain Olfactory Sensory Neuron Synaptic Connectivity in an Early-Life Critical Period

Early-life olfactory sensory experience induces dramatic synaptic glomeruli remodeling in the Drosophila juvenile brain, which is experientially dose-dependent, temporally restricted, and transiently reversible only in a short, well-defined critical period. The directionality of brain circuit synaptic connectivity remodeling is determined by the specific odorant acting on the respondent receptor class of olfactory sensory neurons. In general, each neuron class expresses only a single odorant receptor and innervates a single olfactory synaptic glomerulus. In the Drosophila genetic model, the full array of olfactory glomeruli has been precisely mapped by odorant responsiveness and behavioral output. Ethyl butyrate (EB) odorant activates Or42a receptor neurons innervating the VM7 glomerulus. During the early-life critical period, EB experience drives dose-dependent synapse elimination in the Or42a olfactory sensory neurons. Timed periods of dosed EB odorant exposure allow investigation of experience-dependent circuit connectivity pruning in juvenile brain. Confocal microscopy imaging of antennal lobe synaptic glomeruli is done with Or42a receptor-driven transgenic markers that provide quantification of synapse number and innervation volume. The sophisticated Drosophila genetic toolkit enables the systematic dissection of the cellular and molecular mechanisms mediating brain circuit remodeling.

<meta charset="utf8"></head><body>作者聚焦:探索关键时期经验依赖性突触修剪中的神经胶质影响

We describe here methods for inducing and analyzing olfactory experience-dependent remodeling of antennal lobe synaptic glomeruli in the Drosophila juvenile brain.

果蝇分子和免疫组化分析制备周围嗅组织的研制

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

协议的可视化类固醇机关及其互动器官在果蝇免疫染色

免疫组化标记在感觉神经元树突的微管，气管，并在肌肉果蝇幼虫体壁

嗜酸性粒植物视网膜免疫组织化学、西方分析和 Rna 分离的解剖

嗅觉受体神经元在全山免疫标记

成人的制备果蝇的眼睛

电生理记录

早期关键时期少年脑嗅觉感觉神经元突触连接的经验依赖性重塑

早期关键时期少年脑嗅觉感觉神经元突触连接的经验依赖性重塑

早期关键时期少年脑嗅觉感觉神经元突触连接的经验依赖性重塑