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。

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			<td height="21" style="height:21px;width:271px;">10X Phosphate-buffered Saline</td>
			<td style="width:236px;">Gibco</td>
			<td style="width:241px;">70011-044</td>
			<td style="width:432px;">Dilute to 1X in RNase free water</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CO2 tank</td>
			<td>Air Gas</td>
			<td>CD R200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Two sharp forceps (Dumont #55)</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trizol</td>
			<td>Invitrogen</td>
			<td>15596-026</td>
			<td>RNA isolation solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small bucket with ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">P20 and P200 micropipettes and tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.7 mL Microfuge tubes</td>
			<td></td>
			<td></td>
			<td>Purchase nuclease free for best results</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2 mL PCR tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde powder</td>
			<td>Polysciences</td>
			<td>380</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10% Triton X-100</td>
			<td>Teknova</td>
			<td>T1105</td>
			<td>Dilute to 0.2% v/v in 1X PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2&#34; petri dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">25 C incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">deionized H2O</td>
			<td></td>
			<td></td>
			<td>Purchase nuclease free or treat with DEPC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Rabbit anti-GFP antibody</td>
			<td>MBL international corpor</td>
			<td>PM005</td>
			<td>primary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse anti RAT CD2</td>
			<td>AbD seroTec</td>
			<td>MCA154RHDL</td>
			<td>primary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ADL195 (anti lamin, mouse)</td>
			<td>Dev studies hydoma ban</td>
			<td>ADL195-s</td>
			<td>primary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor&#174; 488 goat anti-rabbit IgG (H+L)</td>
			<td>invitrogen</td>
			<td>A11008</td>
			<td>Secondary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cy3 Goat Anti-Mouse IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-166-003</td>
			<td>Secondary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100, Protein Grade Detergent, 10% Solution, Sterile-Filtered</td>
			<td>CALBIOCHEM</td>
			<td>648463</td>
			<td>detergent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lab Rotator</td>
			<td>Thermo scientific</td>
			<td></td>
			<td>Rotator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nuclease Free Water</td>
			<td>Growcells.com</td>
			<td>NUPW-0125</td>
			<td>Water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light-Duty Tissue Wipers</td>
			<td>VWR</td>
			<td>82003-822</td>
			<td>For cleaning dissection supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superscript II</td>
			<td>invitrogen</td>
			<td>18064014</td>
			<td>Reverse Transcriptase&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAshredder (50)</td>
			<td>RNA extraction</td>
			<td>QIAGEN</td>
			<td>79654</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oligo(dT)12-18 Primer</td>
			<td>RT</td>
			<td>life technologies</td>
			<td>18418012</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNeasy MinElute Cleanup Kit (50)</td>
			<td>RNA extraction</td>
			<td>QIAGEN</td>
			<td>74204</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FASTSTART UNIV SG MASTER (5 ML)(500 RXN)</td>
			<td>qPCR</td>
			<td>Roche</td>
			<td>04913850001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FASTSTART ESSENTIAL GREEN DNA MASTER&#160;</td>
			<td>qPCR</td>
			<td>Roche</td>
			<td>06402712001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIGHTCYCLER 480 - MULTIWELL PLATE 96</td>
			<td>qPCR</td>
			<td>Roche</td>
			<td>04729692001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often compromise the lives of those who suffer from these complex diseases. Detecting mitochondrial metabolic changes is vital to the understanding of mitochondrial disorders and mitochondrial responses to pharmacological agents. Although mitochondrial metabolism is at the core of metabolic regulation, the detection of subtle changes in mitochondrial metabolism may be hindered by the overrepresentation of other cytosolic metabolites obtained using whole organism or whole tissue extractions. 
Here we describe an isolation method that detected pronounced mitochondrial metabolic changes in Drosophila that were distinct between whole-fly and mitochondrial enriched preparations. To illustrate the sensitivity of this method, we used a set of Drosophila harboring genetically diverse mitochondrial DNAs (mtDNA) and exposed them to the drug rapamycin. Using this method we showed that rapamycin modifies mitochondrial metabolism in a mitochondrial-genotype-dependent manner. However, these changes are much more distinct in metabolomics studies when metabolites were extracted from mitochondrial enriched fractions. In contrast, whole tissue extracts only detected metabolic changes mediated by the drug rapamycin independently of mtDNAs.

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Mitochondria play central roles in the regulation of metabolism and homeostasis. Subtle changes in mitochondrial metabolism that affect organismal physiology could be difficult to detect in whole organism metabolomics studies. Here we describe an isolation method that enhances the detection of subtle metabolic shifts in Drosophila melanogaster.

在代谢分析准备线粒体富集级的<em&gt;果蝇</em

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often ...

科研

发育生物学

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.

斑马鱼视网膜蛋白定位的相关超分辨和电镜观察

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

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 ...

A Quantitative Measurement of Reactive Oxygen Species and Senescence-associated Secretory Phenotype in Normal Human Fibroblasts During Oncogene-induced Senescence

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. Recent studies have extended the role of cellular senescence to various physiological processes, including development, wound healing, immune surveillance, and age-related tissue dysfunction. Although cell cycle arrest is a critical hallmark of cellular senescence, an increased intracellular reactive oxygen species (ROS) production has also been demonstrated to play an important role in the induction of cellular senescence. In addition, recent studies revealed that senescent cells exhibit potent paracrine activities on neighboring cells and tissues through a senescence-associated secretory phenotype (SASP). The sharp increase in interest regarding therapeutic strategies against cellular senescence emphasizes the need for a precise understanding of senescence mechanisms, including intracellular ROS and the SASP. Here, we describe protocols for quantitatively assessing intracellular ROS levels during H-Ras-induced cellular senescence using ROS-sensitive fluorescent dye and flow cytometry. In addition, we introduce sensitive techniques for the analysis of the induction of mRNA expression and secretion of SASP factors. These protocols can be applied to various cellular senescence models.

Intracellular ROS has been shown to play an important role in the induction of cellular senescence. Here, we describe a sensitive assay for quantifying ROS levels during cellular senescence. We also provide protocols for assessing the senescence-associated secretory phenotype, which reportedly contributes to various age-related dysfunctions.

基因型衰老过程中正常人成纤维细胞活性氧和衰老相关分泌表型的定量测定

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. ...

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of cells or tissues in model organisms. These systems contain two components as separate transgenic lines. A driver line expresses a transcriptional activator under the control of tissue-specific promoters/enhancers, and a reporter/effector line harbors a target gene placed downstream to the binding site of the transcription activator. Animals harboring both components induce tissue-specific transactivation of a target gene expression. Precise spatiotemporal expression of the gene in targeted tissues is critical for unbiased interpretation of cell/gene activity. Therefore, developing a method for generating exclusive cell/tissue-specific driver lines is essential. Here we present a method to generate highly tissue-specific targeted expression system by employing a &#34;Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated&#34; (CRISPR/Cas)-based genome editing technique. In this method, the endonuclease Cas9 is targeted by two chimeric guide RNAs (gRNA) to specific sites in the first coding exon of a gene in the Drosophila genome to create double-strand breaks (DSB). Subsequently, using an exogenous donor plasmid containing the transactivator sequence, the cell-autonomous repair machinery enables homology-directed repair (HDR) of the DSB, resulting in precise deletion and replacement of the exon with the transactivator sequence. The knocked-in transactivator is expressed exclusively in cells where the cis-regulatory elements of the replaced gene are functional. The detailed step-by-step protocol presented here for generating a binary transcriptional driver expressed in Drosophila fgf/branchless-producing epithelial/neuronal cells can be adopted for any gene- or tissue-specific expression.

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

Here, we present a method for generating tissue-specific binary transcription systems in Drosophila by replacing the first coding exon of genes with transcription drivers. The CRISPR/Cas9-based method places a transactivator sequence under the endogenous regulation of a replaced gene, and consequently facilitates transctivator expression exclusively in gene-specific spatiotemporal patterns.

基于基因组编辑的果蝇组织特异二进制转录系统的高效生成策略

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of ...

Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a variety of human retinal degenerative diseases and pinpoint new treatment approaches. The nuclear architecture of mouse retina is organized in two different patterns: conventional and inverted. Conventional pattern is universal where heterochromatin is localized to the periphery of the nucleus, while active euchromatin resides in the nuclear interior. In contrast, inverted nuclear pattern is unique to the adult rod photoreceptor cell nuclei where heterochromatin localizes to the nuclear center, and euchromatin resides in the nuclear periphery. DNA methylation is predominantly observed in chromocenters. DNA methylation is a dynamic covalent modification on the cytosine residues (5-methylcytosine, 5mC) of CpG dinucleotides that are enriched in the promoter regions of many genes. Three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) participate in methylation of DNA during development. Detecting 5mC with immunohistochemical techniques is very challenging, contributing to variability in results, as all DNA bases including 5mC modified bases are hidden within the double-stranded DNA helix. However, detailed delineation of 5mC distribution during development is very informative. Here, we describe a reproducible technique for robust immunohistochemical detection of 5mC and another epigenetic DNA marker 5-hydroxymethylcytosine (5hmC), which colocalizes with the &#34;open&#34;, transcriptionally active chromatin in developing and postmitotic mouse retina.

The objective of this report is to describe the protocol for robust immunohistochemical detection of epigenetic markers, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing and postmitotic mouse retina.

5-甲基胞嘧啶和 5-Hydroxymethylcytosine 在小鼠视网膜发育和分裂后中的免疫组化检测

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a ...

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ellipsoid shape during development. This developmental process is called oogenesis and is crucial to generating functional mature eggs to secure the next fly generation. For these reasons, egg chambers have served as an ideal and relevant model to understand animal organ development.
Several in vitro culturing protocols have been developed, but there are several disadvantages to these protocols. One involves the application of various covers that exert an artificial pressure on the imaged egg chambers in order to immobilize them and to increase the imaged acquisition plane of the circumferential surface of the analyzed egg chambers. Such an approach may negatively influence the behavior of the thin actomyosin machinery that generates the power to rotate egg chambers around their longer axis.
Thus, to overcome this limitation, we culture Drosophila egg chambers freely in the media in order to reliably analyze actomyosin machinery along the circumference of egg chambers. In the first part of the protocol, we provide a manual detailing how to analyze the actomyosin machinery in a limited acquisition plane at the local cellular scale (up to 15 cells). In the second part of the protocol, we provide users with a new Fiji-based plugin that allows the simple extraction of a defined thin layer of the egg chambers&#8217; circumferential surface. The following protocol then describes how to analyze actomyosin signals at the tissue scale (&#62;50 cells). Finally, we pinpoint the limitations of these approaches at both the local cellular and tissue scales and discuss its potential future development and possible applications.

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

This protocol provides a Fiji-based, user-friendly methodology along with straightforward instructions explaining how to reliably analyze actomyosin behavior in individual cells and curved epithelial tissues. No programming skills are required to follow the tutorial; all steps are performed in a semi-interactive manner using the graphical user interface of Fiji and associated plugins.

使用培养的果蝇卵室延时电影对局部细胞和组织尺度的actomyosin动力学的分析

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

解冻、培养和冷冻保存的嗜酸性粒细胞系

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...