我们感谢熊莹博士对手稿的讨论和评论。这项工作得到了中国国家科学基金委员会（NSFC）对C.M.（31500839）的资助。

1.幼虫解剖
注意：解剖溶液血淋巴样盐水 （HL3.1）18 和固定溶液 4% 多聚甲醛 （PFA）19,20 在室温下使用，因为当温度过低时微管会解聚。
<ol><li>用长钝镊子挑选一只流浪的 3龄 幼虫。用HL3.1洗涤，并将其放在立体显微镜下的解剖皿上。 注：徘徊的3龄 幼虫由分枝的前螺旋体识别，并在约96小时（25°C）21时在管周围爬行。</li>
	<li>将幼虫的背侧或腹侧朝上放置。通过两个长气管识别背侧，通过腹部齿带识别腹侧。<ol><li>根据要观察的组织，选择背侧或腹侧解剖。这将允许更精确、更详细地了解感兴趣的特定区域。对于NMJ和肌肉细胞观察，分别切开幼虫背侧和腹侧区域。</li>
	</ol></li>
	<li>别住嘴钩和尾巴。调整销钉以保持幼虫处于伸展状态。在幼虫中加入一滴HL3.1，以防止其干燥。 注意：不含 Ca2+ 的 HL 3.1 可用于最大限度地减少解剖过程中肌肉的收缩。</li>
	<li>用解剖剪刀在靠近后端的地方做一个小的横向切口，但不要剪掉后端。然后沿腹侧中线向前端切开。</li>
	<li>在幼虫的四个角上插入四个昆虫针。重新调整昆虫针，使幼虫在各个方向上最大限度地伸展。</li>
	<li>使用镊子切除内脏，同时不损伤肌肉。</li>
</ol>2. 固定
<ol><li>加入 100 μL PFA （4%） 将胴体浸泡 40 分钟，同时胴体仍固定在解剖皿上。 注意： 采取有效的保护措施以避免直接接触皮肤或吸入，因为 PFA 是一种危险。</li>
	<li>卸下针脚并将样品转移到 2 mL 微量离心管中。用含有0.2%Triton X-100（0.2%PBST）的1x磷酸盐缓冲盐水冲洗PFA，以15rpm在脱色振荡器上填充2mL微量离心管10分钟。重复洗涤过程 5 次。</li>
</ol>3. 免疫细胞化学
<ol><li>将胴体浸入封闭剂（5%山羊血清在0.2%PBST中）中，并在室温下封闭40分钟。</li>
	<li>除去封闭剂，并用200μL的一抗（例如，抗α-微管蛋白，1：1000;抗Futsch，1：50）在4°C下用0.2%PBST稀释过夜。通过用单克隆抗α-微管蛋白免疫染色来观察肌肉微管。Anti-Futsch可以间接反映NMJ中的微管形态。</li>
	<li>一抗孵育后，用0.2%PBST洗涤幼虫10分钟，重复5次。</li>
	<li>将幼虫与200μL二抗（例如，山羊抗Mouse-488,1：1000）在室温下用0.2%PBST稀释在黑暗中孵育1.5小时。</li>
	<li>接下来，在黑暗20,22中对微管进行染色时，将浓度为1：1000的核染料如TO-PRO（R）3碘化物（T3605）加入孵育管中30分钟。</li>
	<li>用 0.2% PBST 冲洗掉二抗和核染料 10 分钟。在黑暗中重复 5 次。</li>
</ol>4. 安装
<ol><li>将幼虫胴体置于载玻片上的0.2%PBST中，并在立体显微镜下进行调整。确保幼虫胴体的内表面朝上，并且所有幼虫胴体都按所需排列。</li>
	<li>用擦拭物吸收多余的PBST溶液，然后轻轻加入一滴抗淬灭封固剂。</li>
	<li>在载玻片上放置盖玻片，缓慢而轻柔地覆盖解剖的幼虫，以避免气泡。</li>
	<li>在盖玻片周围涂抹指甲油。将载玻片放在黑暗空间中以减少荧光衰减。</li>
</ol>5. 图像采集
<ol><li>要获取图像，请使用激光扫描共聚焦显微镜，选择60倍油浸物镜（数值孔径1.42）或类似物镜，并根据实验调整激光功率和波长。</li>
	<li>要识别 NMJ，请在 A3 段的肌肉 4 中捕获图像（如图 1F 中的位置所示）。选择 488 nm 激光以激活 α-微管蛋白或 Futsch，选择 543 nm 以激活 HRP 成像轨迹。将参数调整为 800 像素 x 800 像素的帧大小、2.0 的数码变焦和 0.8 μm 的成像间隔（图 2）。</li>
	<li>为了识别肌肉中的微管，捕获A3-A5段中肌肉2的图像（ 如图1G中的位置所示），因为它具有较少的气管分支。选择 488 nm 激光器激活 α-微管蛋白，选择 635 nm 激光器激活 T3605 成像轨道。将参数调整为1024像素x 1024像素的帧大小，3.0的数码变焦和肌肉细胞中0.4μm的成像间隔（图3）。</li>
</ol>

观察 果蝇 神经肌肉接头和肌肉细胞中的微管网络

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作者没有什么可透露的。

这里描述了用于 果蝇 幼虫神经肌肉接头和肌肉细胞的解剖和免疫染色的方案。有几个要点需要考虑。首先，在解剖过程中，避免观察到的肌肉受伤至关重要。在切除内脏之前，可能值得固定鱼角，以防止镊子和肌肉直接接触。为避免肌肉损伤或与幼虫表皮分离，重要的是要确保在洗涤和孵育过程中振荡器的速度不超过 15 rpm。此外，必须精确控制皮肤延伸，以确保清晰、完整地拍摄 NMJ 形?...

微管（MTs）作为细胞骨架的结构成分之一，在细胞分裂、细胞生长和运动、细胞内运输和维持细胞形状等多种生物过程中发挥着重要作用。微管动力学和功能通过与其他蛋白质的相互作用进行调节，例如 MAP1、MAP2、Tau、Katanin 和驱动蛋白 1,2,3,4,5。
在神经元中，微管对于轴突和树突的发育和维持至关重要。微管异常会导致功能障碍，甚至导致神经元死亡。例如，在阿尔茨海默氏症患者的大脑中，Tau 蛋白过度磷酸化会降低微管网络的稳定性，导致神经系统异常6。因此，检查微管网络将有助于理解神经发育和神经系统疾病的发病机制。
神经肌肉接头（NMJ）是在运动神经元轴突末梢和肌纤维之间形成的外周突触，是研究突触结构和功能的优秀而强大的模型系统7。Futsch 是果蝇中的一种蛋白质，与哺乳动物中发现的微管结合蛋白 MAP1B 同源8。它仅在神经元中表达，并在 NMJ 突触按钮的发育中发挥作用 8,9。在野生型中，通过用抗Futsch进行免疫染色来观察沿NMJ过程中心运行的丝状束。当到达 NMJ 的末端时，该束能够形成由微管组成的环或失去其丝状结构，从而导致弥漫性和点状外观10。微管环与暂停的生长锥有关，这表明微管阵列是稳定的11。因此，我们可以通过 Futsch 染色间接确定 NMJ 中微管的稳定发育。果蝇幼虫中大尺寸的肌肉细胞可以清晰地观察微管网络。通过分析微管的密度和形状，可以发现影响微管网络稳定性的因素。同时，可以将肌肉细胞的微管网络状态与NMJ的结果进行交叉验证，以获得更全面的结论。
许多协议已被用于研究微管的网络和动力学。然而，这些研究通常集中在体外研究12,13,14,15,16。或者，一些体内实验已经使用电子显微镜来检测细胞骨架17。根据荧光标记的抗体或化学染料与蛋白质或DNA的特异性结合，这里介绍的方法允许在体内单个神经元水平上检测NMJ中的微管网络，结果通过肌肉细胞的观察得到证实。当与果蝇黑腹果蝇中可用的强大遗传工具结合使用时，该方案是简单、稳定和可重复的，能够对体内神经系统中微管网络调节蛋白的作用进行各种表型检查和遗传筛选。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor Plus 405 phalloidin</td>
			<td>invitrogen</td>
			<td>A30104</td>
			<td>dilute 1:200</td>
		</tr>
		<tr>
			<td>Enhanced Antifade Mounting Medium</td>
			<td>Beyotime</td>
			<td>P0128M</td>
			<td></td>
		</tr>
		<tr>
			<td>FV10-ASW confocal microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse antibody, Alexa Fluor 488 conjugated</td>
			<td>Thermo Fisher</td>
			<td>A-11001</td>
			<td>dilute 1:1,000</td>
		</tr>
		<tr>
			<td>Laser confocal microscope LSM 710</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Scissors</td>
			<td>66vision</td>
			<td>54138B</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-Futsch antibody</td>
			<td>Developmental Studies Hybridoma Bank&#160;&#160;</td>
			<td>22C10</td>
			<td>dilute 1:50</td>
		</tr>
		<tr>
			<td>Mouse anti-&#945;-tubulin antibody</td>
			<td>Sigma</td>
			<td>T5168</td>
			<td>dilute 1:1,000</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Wako</td>
			<td>168-20955</td>
			<td>Final concentration: 4% in PB Buffer</td>
		</tr>
		<tr>
			<td>Stainless Steel Minutien Pins</td>
			<td>Entomoravia</td>
			<td></td>
			<td>0.1mm Diam</td>
		</tr>
		<tr>
			<td>Stereomicroscope SMZ161</td>
			<td>Motic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>stereoscopic fluorescence microscope BX41</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Texas Red-conjugated goat anti-HRP</td>
			<td>Jackson ImmunoResearch</td>
			<td></td>
			<td>dilute 1:100</td>
		</tr>
		<tr>
			<td>TO-PRO(R) 3 iodide</td>
			<td>Invitrogen</td>
			<td>T3605</td>
			<td>dilute 1:1,000</td>
		</tr>
		<tr>
			<td>Transfer decoloring shaker TS-8</td>
			<td>Kylin-Bell lab instruments</td>
			<td>E0018</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>BioFroxx</td>
			<td>1139</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers&#160;</td>
			<td>dumont</td>
			<td>500342</td>
			<td></td>
		</tr>
	</tbody>
</table>

using drosophila larval neuromuscular junction and muscle cells to visualize microtubule network

微管网络是神经系统的重要组成部分。许多微管调节蛋白的突变与神经发育障碍和神经系统疾病有关，如微管相关蛋白Tau对神经退行性疾病，微管切断蛋白Spastin和Katanin 60分别引起遗传性痉挛性截瘫和神经发育异常。检测神经元中的微管网络有利于阐明神经系统疾病的发病机制。然而，神经元的小尺寸和轴突微管束的密集排列使得微管网络的可视化具有挑战性。在这项研究中，我们描述了一种解剖幼虫神经肌肉接头和肌肉细胞的方法，以及α-微管蛋白和微管相关蛋白Futsch的免疫染色，以可视化果蝇中的微管网络。神经肌肉接头使我们能够观察突触前和突触后的微管，果蝇幼虫中大尺寸的肌肉细胞可以清晰地观察微管网络。在这里，通过在黑腹果蝇中突变和过表达Katanin 60，然后检查神经肌肉接头和肌肉细胞中的微管网络，我们准确地揭示了Katanin 60在神经发育中的调节作用。因此，结合果蝇黑腹果蝇强大的遗传工具，该方案极大地促进了微管网络调节蛋白在神经系统中的作用的遗传筛选和微管动力学分析。

Microtubules (MTs), as one of the structural components of the cytoskeleton, play an important role in diverse biological processes, including cell division, cell growth and motility, intracellular transport, and the maintenance of cell shape. Microtubule dynamics and function are modulated by interactions with other proteins, such as MAP1, MAP2, Tau, Katanin, and Kinesin1,2,3,4,5.
In neurons, microtubules are essential for the development and maintenance of axons and dendrites. Abnormalities in microtubules lead to dysfunction and even the death of neurons. For instance, in the brain of Alzheimer&#39;s patients, Tau protein hyperphosphorylation reduces the stability of the microtubule network, causing neurological irregularities6. Thus, examining microtubule networks will contribute to a comprehension of neurodevelopment and the pathogenesis of neurological diseases.
The neuromuscular junction (NMJ) is the peripheral synapse formed between a motor neuron axon terminal and a muscle fiber, which is an excellent and powerful model system for studying synaptic structure and functions7. Futsch is a protein in Drosophila that is homologous to the microtubule-binding protein MAP1B found in mammals8. It is expressed only in neurons and plays a role in the development of the NMJ&#39;s synaptic buttons8,9. In wild-type, filamentous bundles that run along the center of NMJ processes are visualized by immunostaining with anti-Futsch. When reaching NMJ&#39;s end, this bundle has the ability to either form a loop consisting of microtubules or to lose its filamentous structure, resulting in a diffuse and punctate appearance10. Microtubule loops are associated with paused growth cones, which suggests the microtubule array is stable11. Therefore, we can indirectly determine the stable microtubule development in NMJ by Futsch staining. The large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. The factors affecting the stability of the microtubule network can be found by analyzing the density and shape of microtubules. Simultaneously, the microtubule network status of muscle cells can be cross-verified with the result of NMJ to obtain more comprehensive conclusions.
Many protocols have been employed for investigating the network and dynamics of microtubules. However, these researches have often focused on in vitro studies12,13,14,15,16. Alternatively, some in vivo experiments have employed electron microscopy to detect the cytoskeleton17. According to the specific binding of fluorescently labeled antibodies or chemical dyes to proteins or DNA, the methods presented here allow the detection of microtubule networks in NMJ at the level of individual neurons in vivo, with results corroborated by observations in muscle cells. This protocol is simple, stable, and repeatable when combined with the powerful genetic tools available in Drosophila melanogaster, enabling a diverse range of phenotypic examinations and genetic screenings for the role of microtubule network regulatory proteins in the nervous system in vivo.

作者聚焦：了解果蝇神经肌肉接头中的微管网络 

使用 果蝇 幼虫神经肌肉接头和肌肉细胞可视化微管网络

Currently, microtubule dynamics are observed indirectly through microtubule-binding proteins such as EB1. Since alphabeta tubulin heterodimers are abundant in the cytoplasm, fold directly, neighboring tubulin heterodimers are lacking the dynamics of microtubule networks in vivo. Our protocol visualizes microtubules in neuromuscular synapses and muscle cells.When combined with the powerful genetic tools available in Drosophila melanogaster, enabling a diverse range of phenotypic examinations and the genetic screening for the role of microtubule network regulatory proteins in the nervous system in vivo. Our research focuses on the relationship between the cytoskeleton and the neurodevelopment, as well as its implications for neurological diseases. We hope to discover the mechanism of neurodevelopment and the parthenogenesis for neurological diseases by highlighting the effect of cytoskeletal regulatory proteins on neurons.

我们演示了可视化神经肌肉接头 （NMJ） 和肌肉细胞中微管网络的分步程序。根据示意图（图1A-E）解剖后，进行免疫染色，随后在激光共聚焦显微镜或立体荧光显微镜下观察和收集图像（图1F，G）。
NMJ的突触前后微管组织都可以用抗α微管蛋白标记，通过选择激光扫描共聚焦显微镜的层厚，可以显示不同?...

Watch this Scientific Journal Video about Understanding Microtubule Network in Drosophila Neuromuscular Junctions at JoVE.com

在这里，我们提出了一个详细的方案来可视化神经肌肉接头和肌肉细胞中的微管网络。结合 黑腹果蝇强大的遗传工具，该方案极大地促进了微管网络调节蛋白在神经系统中的作用的遗传筛选和微管动力学分析。

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with ...

目前，微管动力学是通过微管结合蛋白（如EB1）间接观察到的。由于微管蛋白异二聚体在细胞质中含量丰富，直接折叠，相邻的微管蛋白异二聚体缺乏体内微管网络的动力学。我们的方案可视化了神经肌肉突触和肌肉细胞中的微管。当与黑腹果蝇中可用的强大遗传工具相结合时，可以进行各种表型检查和遗传筛选，以发现微管网络调节蛋白在体内神经系统中的作用。我们的研究重点是细胞骨架与神经发育之间的关系，以及它对神经系统疾病的影响。我们希望通过强调细胞骨架调节蛋白对神经元的影响来发现神经发育和神经系统疾病孤雌生殖的机制。

using-drosophila-larval-neuromuscular-junction-muscle-cells-to

Research

JoVE Journal

Neuroscience

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

1.4K 次观看.  Hubei University. 目前，微管动力学是通过微管结合蛋白（如EB1）间接观察到的。由于微管蛋白异二聚体在细胞质中含量丰富，直接折叠，相邻的微管蛋白异二聚体缺乏体内微管网络的动力学。我们的方案可视化了神经肌肉突触和肌肉细胞中的微管。当与黑腹果蝇中可用的强大遗传工具相结合时，可以进行各种表型检查和遗传筛选，以发现微管网络调节蛋白在体内神经系统中的作用。我?...

视频: 作者聚焦：了解果蝇神经肌肉接头中的微管网络 

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of &#945;-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

科研

神经科学

Drosophila Larval Dissection and Fixation for Microtubule Visualization

Begin by picking out a wandering Drosophila third instar larva using long blunt forceps. Wash the larva with hemolymph-like saline, then dry it with a wipe. Next, position the larva dorsally or ventrally on a dissection dish under the stereo microscope.Depending on the tissue to be observed, opt for dorsal or ventral dissection. Pin the larva's mouth hooks and tail to maintain extended positioning. Next, add a drop of the hemolymph-like saline to prevent the larva from drying.Using dissection scissors, make a small transverse incision close to the posterior end without severing it completely. Proceed to cut along the ventral midline, moving toward the anterior end. Now insert four insect pins each into the corners of the larva.Make necessary adjustments to the insect pins to maximize the stretching of the larva. Use forceps to gently remove the internal organs while avoiding harm to the muscles. To fix the larva, immerse the carcasses in 100 L of 4%paraformaldehyde, while still affixed to the dissecting dish.After this, dismount the pins. Then transfer the sample into a 2 mL microcentrifuge tube filled with 0.2%PBST. Place the tube on a shaker for 10 minutes at 15 RPM.

果蝇 用于微管可视化的幼虫解剖和固定

Immunohistochemistry of Drosophila Larva for Microtubule Visualization

Remove the PBST and immerse the paraformaldehyde-fixed drosophila larval carcasses in a blocking agent for 40 minutes at room temperature. Replace the blocking agent with 200 microliters of the primary antibody diluted with 0.2%PBST. Leave the larvae to incubate at four degrees Celsius overnight.The next day, wash the larvae with 0.2%PBST for 10 minutes. Next, remove the PBST from the tube and add 200 microliters of diluted secondary antibody. Incubate it at ambient temperature for 1.5 hours in the dark.Introduce a nuclear dye such as TO-PRO R 3 iodide into the incubating tube for 30 minutes while staining the microtubules in the dark. Finally, rinse off the secondary antibody and the nuclear dye with 0.2%PBST for 10 minutes.

果蝇幼虫的免疫组织化学用于微管可视化

Mounting and Image Acquisition of Drosophila Larval Microtubules

Begin by placing the antibody labeled drosophila larval carcasses in 0.2%PBST on a glass slide. Adjusted under the stereo microscope, ensuring that the inner surface of the larval carcass is facing up. Absorb the excess PBST solution using a wipe.Then delicately add a drop of Anti-Fade mounting medium. Next place a cover slip on the slide covering the dissected larvae slowly and gently, avoiding bubble formation. Secure the cover slip in place by applying fingernail polish around its edges.Store the slide in a dark location to minimize fluorescent attenuation. For image acquisition, use the laser scanning confocal microscope with the 63x Oil immersion objective. Then adjust the wavelength and laser power to fit the requirements of the experiment best.To identify the neuromuscular junctions and capture images of muscle four in segment A three, select a 488 nanometer laser to activate alpha tubulin or Futsch, and 546 nanometers to activate the HRP imaging track. Modify the parameters to achieve a frame size of 1, 024 by 1, 024 pixels, a digital zoom of 1.0, and an imaging interval of 0.8 micrometers. To visualize microtubules in the muscle, capture images of muscle two in the segments A three to A five as it has fewer tracheal branches.Choose a 488 nanometer laser for activating alpha tubulin and a 633 nanometer laser for activating the T3605 imaging track. Adjust the imaging parameters to a frame size of 1, 024 by 1, 024 pixels, a digital zoom of 2.0, and an imaging interval of 0.4 micrometers. Both pre-and post-synaptic microtubule organizations of the neuromuscular junction were labeled with anti-alpha tubulin.Futsch staining reflected the abundance of stable microtubules in the pre-synaptic neurons. Decreased signal intensity of anti-Futsch was observed when microtubules severing protein Katanin 60 was overexpressed in the presynaptic neurons. The staining intensity of the axon trunk was stronger than that of the branches.Katanin 60 mutations resulted in increased microtubule loops. Overexpression of Katanin 60 caused short microtubule fragments within the terminal buttons. Alpha tubulin staining demonstrated a clear network of microtubules around the nucleus.Muscle cells had a significantly increased perinuclear microtubular intensity and exhibited stronger bundles in the Katanin 60 mutant. Overexpression resulted in fragmentation of the microtubule fibers.