Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection
Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection
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DOI：
10.3791/3080
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发表时间：
2011-06-01
影响因子：
1.2
通讯作者：
Godenschwege, Tanja A.
中科院分区：
文献类型：
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作者：
Boerner, Jana;Godenschwege, Tanja A.
为了分析神经元的轴突和树突形态，获得神经元结构的准确标记是必不可少的。制备标记良好的样本，几乎没有组织损伤，使我们能够分析细胞形态，并将单个样本相互比较，从而识别突变异常。在演示的解剖方法中，神经系统主要保留在成年果蝇体内。通过背部切口，打开腹部和胸部，取出大部分内脏。只有腹神经索（VNC）的背侧和含有巨纤维（GF）的大轴突的颈结缔组织（CvC）暴露（1），而含有GF细胞体和树突的脑保留（2）在完整的头部中。在这个标本中，VNC的大部分神经应该保持与肌肉的连接。解剖后，显示了荧光染料填充的巨纤维（GF）的细胞内。在CvC中，GF轴突位于背表面，因此可以在具有微分干涉对比（DIC）光学器件的显微镜下容易地可视化。这允许在该位点用染料注射GF轴突以标记整个GF，包括VNC中的轴突及其末端。该方法导致GF的可靠和强染色，允许神经元在用落射荧光显微镜填充后立即成像。或者，可以使用适用于高分辨率共聚焦显微镜的标准免疫组织化学程序（3）来增强荧光信号。
To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective (CvC) containing the big axons of the giant fibers (GFs)(1) are exposed, while the brain containing the GF cell body and dendrites remains(2) in the intact head. In this preparation most nerves of the VNC should remain attached to their muscles.Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures(3) suitable for high resolution confocal microscopy.