作者感谢翻译基因组小组成员和凯瑟琳·汉森的反馈和对该协议的改进。这个项目是与研究赠款 SAF2015-64500-R, 其中包括欧洲区域发展基金, 授予 R. A 由 Ministerio de Economia y Competitividad。

1. 固定和树脂嵌入
<ol><li>麻醉与二氧化碳 (CO2) 或通过低温使用冰块的苍蝇。使用解剖显微镜 (用低放大率观察整个苍蝇) 和剪刀, 以消除腿部, 翅膀, 头部和腹部的末端部分, 以促进固定剂的穿透。为了避免胸部的变形, 需要在这一步骤中小心地处理尸体。 注: 每个基因型至少有12只苍蝇, 以确保在手术结束时有足够数量的经过适当处理的个体。</li>
	<li>将尸体转移到冰上的1.5 毫升管上 (每管最多放置6个人), 其中含有200µL 溶液1。 注: 屠体不能在解决方案1中保留超过20分钟。<ol>
<li>对于解决方案 1, 混合组件在这些体积比例: ¼4% 多聚甲醛, ¼8% 戊二醛, ¼0.2 米Na2HPO 4 和¼ 0.2 MNaH2PO4。</li>
	</ol>
</li>
	<li>加入200µL 溶液 2, 在冰上孵育30分钟。<ol>
<li>解决方案 2: 在 1:1 (v/v) 比例中混合解决方案1和 OsO4 。 警告: OsO4非常有毒。使用适当的安全和处置措施在通风罩中操作。</li>
	</ol>
</li>
	<li>除去所有的液体。加入200µL 溶液 2, 在冰上孵育1-2 小时。</li>
	<li>去除溶液, 通过分级乙醇系列脱水样品: 一次在 30%, 50%, 70%, 90%, 和两次在绝对乙醇;每人5分钟。添加1毫升的乙醇来覆盖样品。 注: 一旦将组织放置在90% 乙醇中, 随后的步骤可以在室温下进行。</li>
	<li>在丙烯氧化物中孵化两次样品, 每次孵化10分钟。 注意: 氧化丙烯是有毒的, 用在通风罩里。</li>
	<li>用 1:1 (v/v) 的环氧树脂和环氧丙烷混合物取代丙烯氧化物, 在室温下孵化一夜。 注意: 树脂在液体状态下是极其有毒的。该树脂可作为一套四组件 (a、B、C 和 D) 提供。<ol>
<li>对于环氧树脂, 在一次性烧杯中加入54克树脂 (a)、44.5 克硬化剂 (B)、2.5 克加速器 (C) 和10克增塑剂 (D)。将所有树脂元件组合在通风罩中。将树脂贮存在整除数-20 摄氏度 (长达6月) 或-80 摄氏度 (数年)。</li>
		<li>为了去除树脂, 烘烤它在70°c 为 24 h, 因为固体树脂不是毒物。根据适当的国际程序, 丢弃重金属组中以前的所有解决方案, 因为解决方案可以包含 OsO4 的跟踪.
</li>
	</ol>
</li>
	<li>用100% 树脂取代以前的混合物, 并孵育样品4小时。</li>
	<li>将壳体转移到含有新树脂的塑料模具 (模具中每个井中的一个胴体)。使用削尖的木棒或针转移和定向在水井中的尸体, 并让树脂聚合一夜之间在巴斯德烤箱在摄氏70摄氏度。 注: 为了确保后修剪和切片的胴体的适当方向, 尸体应该躺在他们的长边, 与前部的胴体非常接近井的边缘。</li>
</ol>2. 砌块的修整和切片
<ol><li>从模具中取出聚合树脂块。使用剃刀刀片修剪树脂, 形成一个梯形形状周围的胴体, 并给予适当的方向在切片。开始切片所有的尸体后, 横向缝合, 以获得可比的结果。 注意: 该节必须垂直于 DLMs 的前后轴, 才能正确修剪示例 (图 3)。</li>
</ol><img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56179/56179fig3.jpg"> 图 3.Dorsoventral 部分的树脂嵌入成人胸膛的强直营养不良模型苍蝇.在 (A) 中, 样品的修剪不正确, 因为图像的右背部分的部分不是完全横向的肌肉。因此, 右上角的三块肌肉束似乎比左侧的大。这个错误会导致对肌肉区域的高估。(B) 部分从正确修剪的树脂块。刻度条: 200 µm。
<ol><li>切割 1.5-µm 厚的部分与 ultramicrotome, 并转移到水下降到糊幻灯片的部分。<ol>
<li>从 mesothorax 段获取所有样本的部分, 以获得可比较的结果。</li>
	</ol>
</li>
	<li>将包含节的幻灯片放在加热块上70-80 摄氏度, 直到 H2O 蒸发。此时, 可以使用 OsO4对比度 (图 4A) 来观察示例。</li>
</ol>3. 染色 IFM 部分
<ol><li>盖部分与足够的甲苯胺蓝色大约三十年代在热化块。<ol>
<li>甲苯胺蓝溶液, 溶解1% 甲苯胺蓝和1% 硼砂在 H2O。</li>
	</ol>
</li>
	<li>用 H2进行冲洗, 并将幻灯片放在暖盘上以蒸发水。如果需要增强对比度 (图 4B), 请重复使用甲苯胺蓝染色。</li>
</ol><img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56179/56179fig4.jpg"> 图 4.Dorsoventral 部分树脂嵌入成人胸膛不同对比度染色.两个面板都显示了 DM1 模型苍蝇的图像, 与 Abp1 一样, 如图 2D所示。(a) 带有 OsO4对比度的节的图像。(B) 染色甲苯胺蓝的部分, 可以更好地可视化肌肉束。刻度条: 200 µm。
4. 安装 IFM 部分
<ol><li>用加热块干燥幻灯片, 直到 H2O 蒸发。</li>
	<li>将1或2滴的安装介质放在切片上, 然后放在盖玻片上。 注意: 在通风罩中执行这一步骤, 因为安装介质是有毒的。</li>
</ol>5. 图像的采集和肌肉面积的量化
<ol><li>以低放大率的图像, 使整个 DLM 肌肉集中。我们通常使用10X。<ol>
<li>统计分析每飞至少需要5个序列图像, 每实验组分析至少5只苍蝇。</li>
	</ol>
</li>
	<li>选择包含整个肌肉的图像 (图 5A)。检查该部分的轴或方向, 并丢弃不适当的方向的样本, 这将扭曲结果 (比较图 3/3B, 以区分严重和良好的部分, 分别)。</li>
	<li>使用 ImageJ 软件选择仅包含 DLMs (图 5A/5B) 的区域 (以像素为单位)。为了比较不同的实验组, 要使用的参考区域总是以最大的肌肉飞行。在要比较的不同基因型的所有图像中, 选择包含参照区域 (图 5B) 的相等尺寸 DLMs 的区域。</li>
	<li>Binarize 图像-J 命令:进程/二进制/二进制并删除与感兴趣的肌肉不对应的区域或像素 (图 5C)。<ol>
<li>如果 artefactual 黑点出现在肌肉以外的区域, 请使用命令绘图工具将其删除, 然后选择橡皮擦符号。</li>
	</ol>
</li>
</ol><img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56179/56179fig5.jpg"> 图 5.肌肉面积测定的图像分析程序.(A) 强直营养不良症1型的横断面与含有 DLM 的矩形一起飞行胸部。(B) 选定区域仅有间接飞行肌肉和肠道 (*)。(C) 二值化图像。黑色区域对应于兴趣的肌肉。肠道已被清除, 以准确定量的肌肉面积。刻度条: 200 µm。
<ol start="5"><li>将与肌肉组织 (黑色像素) 对应的像素百分比量化为命令:分析/测量并选择区域分数选项。这个百分比的面积是估计的 DLM 肌肉质量的每一个苍蝇。</li>
</ol>

<ol>
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没有宣布利益冲突。

已证明,果蝇是研究人类神经肌肉疾病的有用模型7,10,11, 包括强直白斑, 其特点是外观肌肉萎缩。这里提出的协议是一个有用的工具, 以量化的肌肉退化造成的发病或进展的某一特定疾病的苍蝇模型。例如, 通过对不同年龄的苍蝇进行这种分析, 也可以追踪和量化肌肉纤维的变性。
该协议中有关键步骤?...

果蝇的胸部包含两种不同种类的飞行肌肉, 它们在功能上、生理上和解剖学上截然不同。这些肌肉是: 间接飞行肌肉 (IFM), 由 dorso 纵向 (DLM) 和 dorso 腹 (DVM) 肌肉 (图 1) 和同步飞行控制肌肉1,2组成。这些肌肉共同产生飞行所需的高机械动力。IFMs 的大小、分布和 rostro 尾部配置允许对横向切片3 (图 2A) 进行简单的定位。因此, 我们选择了这些肌肉来研究果蝇的肌肉萎缩症。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56179/56179fig1.jpg"> 图1。图的胸部的果蝇显示间接飞行肌肉 (IFMs) 安排。(左) 代表侧面视图, (右) 表示胸部的横断面。IFMs 由 Dorso 纵向 (DLM) 肌肉 (红色) 和 Dorso 腹 (DVM) 肌肉 (绿色) 组成。
组织结构的保存和对 dorso-腹侧轴方向的控制是确保正确评估肌肉横截面积的关键。为了保持肌肉结构, 我们使用了从汤姆林森et . 修改的固定混合物。4 。此外, 由于肌肉是内部组织, 因此, 由于固定混合物无法穿透靶组织, 因此,果蝇外骨骼的抗渗性是一个问题。为了规避这个问题, 我们删除了苍蝇头, 腿, 翅膀和最后两个部分的腹部创造孔, 使固定合剂进入。作为固定协议的一部分, 我们包括使用锇毒气 (OsO4)5进行治疗, 因为它能够修复脂肪, 包括甘油三酯, 因此被广泛使用。OsO4保留大多数结构非常好, 特别是在细胞学级别, 同时提供与图像的对比度。固定后,果蝇胸膛嵌入在树脂中进行横向半薄切片 (1.5 µm)。为改善对比, 组织可以另外染色甲苯胺蓝。完全胸膛的图像是在10X 和肌肉区域被量化的 binarizing 图像 (等维度) 和量化百分比的像素对应的肌肉组织 (黑色像素) 的总和, 与 ImageJ 软件。
在本议定书中介绍的 OsO4和戊二醛溶液浓度的增加, 对组织制备和固定混合物的修改, 使肌肉组织得以独特的保存。这是因为该协议避免了组织的退化和变形, 使样品的后分析更可靠, 即使在高度萎缩的情况下与神经肌肉退行性疾病, 如强直营养不良 (DM) 有关。在更常见的形式, DM 类型 1, 这种罕见的遗传紊乱是由扩大 CUG 重复在强直营养不良蛋白激酶(DMPK) 转录。突变 DMPK rna 聚集形成 ribonuclear 病灶, 它能封存 Muscleblind 核 RNA 结合蛋白 (MBNL1-3;果蝇)6中的 Muscleblind。我们通过在肌球蛋白重链启动子 (Mhc-Gal4) 下表达 250 CTG 重复, 生成了一种强直营养不良的果蝇模型。模型苍蝇是不能飞的, 典型的 "上升的翅膀" 表型和严重的肌肉萎缩, 在他们的 IFMs (图 2B)。以前在我们实验室进行的研究表明, IFMs 的肌肉面积的测定是一种可靠的方法来量化不同化学或遗传修饰剂的肌肉萎缩的影响, 这些模型苍蝇7。举例来说, 在飞行中表达 250 CTG 重复的异构体 C 在肌肉中的过度表达, 实现了肌肉区域的抢救, 因为螯合被封存的损耗是 DM1 发病机制8 (图 2C) 的触发因素。肌肉区域也获救后喂养 DM 模型苍蝇与 Abp1, 一个 hexapeptide 与证明 anti-DM1 活动9 (图 2D)。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56179/56179fig2.jpg"> 图2。树脂嵌入成人胸膛 dorsoventral 段的量化。(A D) 间接飞行肌肉的果蝇与指定的相关基因型。(A) 控制苍蝇 (yw)。(B) 在肌肉中250非编码 CTG 重复的表达 (无人参与-ctg (250) x) 导致 DLMs 中肌肉面积的减少与控制苍蝇相比. (C) 这种肌肉萎缩表型是通过过度表达 Muscleblind (MblC) (无人飞机-ctg (250) x 无人) 和 (D) 喂养模型苍蝇与 hexapeptide Abp1 (无人飞机-ctg (250) x Abp1)。在所有图像中, 背侧位于顶部。转基因是用肌球蛋白重链启动子 (Mhc)-Gal4 驱动肌肉。(E) 相对于对照蝇的肌肉面积百分比进行量化, 证实差异很大。直方图显示的意思是 S.E.M.p< 0.01 和 * p < 0.05 (Student´s t 测试)。刻度条: 200 µm.<a href="//cloudflare.jove.com/files/ftp_upload/56179/56179fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
本文所报道的方法将对研究肌肉发育、维持和衰老、疾病病理学和药物检测的研究者们感兴趣, 因为它提供了有关肌肉组织对内源和外在因素的反应的可靠信息。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Image-J software</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>Ultramicrotome</td>
			<td>Leica</td>
			<td>Leica UC6</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td>Leica MZ6</td>
			<td>Bright field technique.</td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>Electron Microscopy Sciences</td>
			<td>71970</td>
			<td>Several alternative providers exist.</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>World Precision World</td>
			<td>14003</td>
			<td>Several alternative providers exist.</td>
		</tr>
		<tr>
			<td>Embedding molds</td>
			<td>Electron Microscopy Sciences</td>
			<td>70900</td>
			<td>Several alternative providers exist.</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Fluka (Sigma)</td>
			<td>49624</td>
			<td>Toxic.</td>
		</tr>
		<tr>
			<td>OsO4</td>
			<td>Polyscience</td>
			<td>0972A</td>
			<td>Extremely toxic.</td>
		</tr>
		<tr>
			<td>Propylene oxide</td>
			<td>Sigma Aldrich</td>
			<td>82320-250ML</td>
			<td>Extremely toxic.</td>
		</tr>
		<tr>
			<td>resin (Durcupan)</td>
			<td>Sigma Aldrich</td>
			<td>44611-44614</td>
			<td>Carcinogenic when it is unpolymerized.</td>
		</tr>
		<tr>
			<td>Toluidine blue</td>
			<td>Panreac</td>
			<td>251176</td>
			<td>Toxic.</td>
		</tr>
		<tr>
			<td>Mountant Medium (DPX)</td>
			<td>Sigma Aldrich</td>
			<td>44581</td>
			<td>Dangerous.</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>P6148-500G</td>
			<td>Harmful.</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Panreac</td>
			<td>122507</td>
			<td>0.2 M dilution.</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Panreac</td>
			<td>121677</td>
			<td>0.2 M dilution.</td>
		</tr>
		<tr>
			<td>Borax</td>
			<td>Panreac</td>
			<td>3052</td>
			<td>Toxic.</td>
		</tr>
	</tbody>
</table>

optical cross-sectional muscle area determination of drosophila melanogaster adult indirect flight muscles

肌肉大量的浪费, 称为肌肉萎缩, 是一个常见的表型在果蝇模型的神经肌肉疾病。我们使用的间接飞行肌肉 (IFMs) 的苍蝇, 特别是 dorso-纵肌 (DLM), 作为实验对象, 以衡量的萎缩表型所带来的不同的遗传原因。在该协议中, 我们描述了如何将飞胸肌嵌入半薄切片, 如何获得良好的肌肉和周围组织的对比, 以及如何处理光学显微镜图像的半自动获取可量化的数据和分析.我们描述方法管道的三具体应用。首先, 我们展示了如何将该方法用于量化强直营养不良蝇模型中的肌肉退化;第二, 肌肉横截面积的测量可以帮助识别促进或防止肌肉萎缩和/或肌肉退化的基因;第三, 该协议可用于确定候选化合物是否能够显著改变由致病突变或环境触发器诱发的特定萎缩表型.

为了量化 MblC 或 Abp1 的过度表达是否对强直营养不良蝇模型的萎缩表型有任何影响, 我们重点关注 DLMs, 这是 IFMs 的一部分 (图 1)。我们确定, 模型苍蝇, 这表明250非编码 CTG 重复整个肌肉组织驱动的肌球蛋白重链启动子 (Mhc)-Gal4, 有一个50% 的减少肌肉面积相比, 控制苍蝇。相反, MblC 和扩张的 CTG 的共同表达与同一驱动程序重复, 强烈抑制这种表型?...

Watch this Scientific Journal Video about Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles at JoVE.com

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

果蝇成人间接飞行肌的光学横截肌面积测定

我们报告的方法, 以量化肌肉面积, 这是一个间接的方法来确定肌肉质量在果蝇成人。我们通过分析强直营养不良症的果蝇模型中的间接飞行肌肉来证明我们的方法的应用。

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight ...

optical-cross-sectional-muscle-area-determination-drosophila

Research

JoVE Journal

Biology

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

8.9K 次观看.  University of Valencia. 我们报告的方法, 以量化肌肉面积, 这是一个间接的方法来确定肌肉质量在果蝇成人。我们通过分析强直营养不良症的果蝇模型中的间接飞行肌肉来证明我们的方法的应用。

视频: Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

在果蝇幼虫中枢神经系统的剖析

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

科研

生物学

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

高分辨率视频跟踪移动的，在成年果蝇。

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Visually Mediated Odor Tracking During Flight in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

在果蝇的飞行过程中可视介导的气味追踪

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

从成人Oenocytes剖析果蝇</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

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

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

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

石蜡包埋及冷冻的部分果蝇成人肌肉

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

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

分离果蝇睾丸

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

注射胰岛素和血淋巴的提取来衡量成人胰岛素敏感性果蝇</em

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

体外评估对离体骨骼肌的收缩，疲劳性和交替"

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

寿命测量<em&gt;果蝇</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Maintenance of a Drosophila melanogaster Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

的维持<em&gt;果蝇</em&gt;人口笼

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...