Boerner Jana, Godenschwege Tanja A
Department of Biological Sciences, Florida Atlantic University, USA.
J Vis Exp. 2011 Jun 4(52):3080. doi: 10.3791/3080.
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) are exposed, while the brain containing the GF cell body and dendrites remains 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 suitable for high resolution confocal microscopy.
为了分析神经元的轴突和树突形态，准确标记神经元结构至关重要。制备标记良好且几乎没有组织损伤的样本，使我们能够分析细胞形态，并将各个样本相互比较，从而识别突变异常。在所示的解剖方法中，神经系统大部分保留在成年果蝇体内。通过背部切口打开腹部和胸部，并移除大部分内部器官。仅暴露腹侧神经索（VNC）的背侧和包含巨纤维（GFs）大轴突的颈神经连索（CvC），而包含GF细胞体和树突的大脑仍保留在完整的头部中。在这种制备方法中，VNC的大多数神经应保持与它们的肌肉相连。解剖后，展示了用荧光染料对巨纤维（GF）进行细胞内填充的过程。在CvC中，GF轴突位于背表面，因此在具有微分干涉对比（DIC）光学系统的显微镜下很容易观察到。这使得可以在该部位向GF轴突注射染料，以标记整个GF，包括其在VNC中的轴突及其末端。这种方法能对GFs进行可靠且强烈的染色，使神经元在用落射荧光显微镜填充后可立即成像。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增强荧光信号。
