优化 果蝇 幼虫神经肌肉接头透射电子显微镜样品制备工艺
Summary
本报告提供了一种新的样品制备程序，用于可视化 果蝇 幼虫中的神经肌肉接头。与传统方法相比，该方法在防止样品卷曲方面更有效，对于 果蝇 神经肌肉接头超微结构分析特别有用。
Abstract
果蝇神经肌肉接头 （NMJ） 已成为神经科学领域有价值的模型系统。在果蝇 NMJ 中应用共聚焦显微镜使研究人员能够获取突触信息，包括突触丰度的定量数据和对其形态的详细见解。然而，透射电镜的漫射分布和有限的视觉范围给超微结构分析带来了挑战。本研究引入了一种创新且高效的样品制备方法，该方法超越了传统方法。该过程首先将金属网放在平底瓶或试管的底部，然后将固定的幼虫样品定位到网状物上。在样品上放置一个额外的网格，确保它们位于两个网格之间。在进行包埋程序之前，将固定样品彻底脱水并渗透。然后以平片方式将样品嵌入环氧树脂中，这允许准备用于定位和切片的肌肉。受益于这些步骤，果蝇幼虫的所有肌肉都可以在光学显微镜下看到，从而促进后续的定位和切片。在定位身体节段 A2 和 A3 的第 6 和第 7块肌肉后，去除多余的树脂。进行第 6或第 7块肌肉的连续超薄切片。
Introduction
电子显微镜是研究生物材料超微结构最理想的方法之一，可以在纳米级1级直观准确地展示细胞的内部结构。然而，由于样品制备过程的复杂性和高昂的成本，电子显微镜不如光学显微镜那么受欢迎。电子显微镜技术的最新进展导致了图像质量的显着提高，同时显着减少了相关的工作量。因此，电子显微镜在推进不同领域的科学知识方面发挥着重要作用2.
果蝇是一种优秀的动物模型，用于执行遗传操作以精确控制靶基因的空间和时间表达3。此外，与哺乳动物模型相比，果蝇具有生长期短、易于饲养的优点;因此，果蝇在形态学研究中被广泛使用4,5。
在果蝇幼虫中，神经肌肉接头 （NMJ） 胸膜广泛分布在肌肉中 6,7，NMJ 的免疫染色可以很容易地提供有关突触数量和形态的信息 8,9 。位于 A2 和 A 3 节段的第 6/7节肌肉中的 NMJ 胸肌非常适合使用光学显微镜进行定量和形态学研究。这是因为它们的大小和丰度10,11。因此，果蝇幼虫 NMJ 被认为是神经科学研究的有用模型12。
然而，通过透射电镜观察NMJ bouton超微结构具有挑战性。由于透射电子显微镜的扫描窗口较窄，因此很难定位广泛分布的NMJ boutons13。另一个原因是，在样品制备方案7的酒精脱水步骤中，果蝇体壁容易卷曲。
传统研究通常选择 A2 和 A 3 节段的第 6 和第7 块肌肉之间的胸针作为样品材料，因为它们的丰度和大小14,15。A2 和 A 3 节段的第 6 和第7 节肌肉比其他肌肉大，并且包含更多的胸花。然而，当制备用于电子显微镜的样品时，固定样品往往会变薄并且容易卷曲，从而导致 A2 和 A3 节的第 6 和第7 块肌肉定位不当。
我们在此报告一种新的处理程序，与传统的样品制备方法相比，该程序更有效地防止样品卷曲7,16，通过允许样品在随后的脱水过程中保持平坦，从而促进果蝇幼虫神经肌肉接头的更好定位。
Protocol
注：本文中使用的透射电子显微镜样品制备方法之前已报道过16.需要注意的是，试剂的选择和剂量的调整是必要的，具体取决于样品。在样品制备过程中使用有毒化学试剂很多，因此，操作人员需要采取一定的防护措施，如穿防护服和手套，在通风橱中操作。 1. 解剖、固定和放置程序 解剖在Jan溶液中解剖游荡的3龄后期幼虫，并根?…
Representative Results
果蝇幼虫体壁由30根可识别的肌肉纤维组成，这些肌肉纤维以规则的模式排列，在解剖和固定后看起来像一片薄片21（图1A）。由于金属网的存在，样品在脱水过程中保持平坦（图1B，C）。将幼虫体肌肉埋在由环氧树脂制成的薄板中（图1D-G），该薄板易于准确地定位并…
Discussion
果蝇 幼虫样品在脱水过程中容易卷曲，因为样品很薄，难以准确定位神经肌肉接头，从而增加了样品制备的难度和工作量。传统的改进是缩短了样品7个，但样品仍进行了不同程度的卷曲。
在我们的方法中，有两个关键步骤：首先，由于金属网的限制，样品在整个脱水过程中保持平坦。此外，在聚合过程中，可以通过将样品夹在塑料制品之间来聚合样…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了中国自然科学基金32070811和东南大学（中国）分析测试基金11240090971的支持。我们感谢中国南京东南大学医学院电子显微镜实验室和形态分析中心。
Materials
| 1,2-Epoxypropane | SHANGHAI LING FENG CHEMICAL REAGENT CO., LTD | JYJ 037-2015 | Penetrating Agent |
| Drosophila Stocks | Bloomington | none | The wild-type control Drosophila strains used in this research were all W1118, and were reared according to standard culture methods |
| Flat-bottomed glass test tubes | Haimen Chenxing Experimental equipment Company | none | Flat-bottomed glass test tubes(bottle)with sponge plug(or bottle stopper) |
| K4M cross-linker | Agar Scientific | Cat# 1924B | The embedding resins are based on a highly cross-linked acrylate and methacrylate formula |
| K4M resin (monomer B) | Agar Scientific | Lot# 631557 | Resin Monomer |
| Polyvinyl film | Haimen Chenxing Experimental equipment Company | none | Transparent polyethylene film is the best , thickness of about 0.2mm |
| SPI Chem DDSA | SPI | SpI#02827-AF | Dodecenyl Succinic Anhydride |
| SPI-Chem DMP-30 Epoxy | SPI | 02823-DA | Accelerator |
| SPI-Chem NMA | SPI | SpI#02828-AF | Hardner for Epoxy |
| SPI-PON 812 Epoxy | SPI | SPI#0259-AB | Resin Monomer |
| Steel mesh | Yuhuiyuan Gardening Store(online) | none | Copper or stainless steel net |
| Transmission electron microscopy | Hitachi H-7650 | 11416692 | All grids (on which samples were gathered) were stained with lead citrate and observed under a transmission electron microscopy. |
| Ultrathin microtome | Leica UC7 ultrathin microtome | 595915 | All sectioning operations are carried out on a Leica UC7 ultrathin microtome using a diamond knife |
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Cite This Article
Guangming, G., Qingyuan, S., Yutong, O., Mei, C., Chenchen, Z. Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae. J. Vis. Exp. (199), e64934, doi:10.3791/64934 (2023).