Hill S J, Oliver D L
Department of Anatomy, University of Connecticut Health Center, Farmington 06030.
J Neurosci Methods. 1993 Jan;46(1):59-68. doi: 10.1016/0165-0270(93)90141-d.
A new method is described that allows identification of neurons by retrograde transport, intracellular injection of biotin-labeled Lucifer Yellow, and a histochemical reaction of the biotin. Cells in the inferior colliculus that project to the ipsilateral thalamus are identified by injection of rhodamine-labeled latex beads into the medial geniculate body and retrograde transport in vivo. Several days later, the brains are fixed with aldehydes, and the inferior colliculus is cut on a vibratome. The rhodamine-labeled cells are observed with epi-fluorescent optics, impaled with intracellular pipettes under visual control, and injected with 9% Lucifer Yellow dilithium salt and 1% Lucifer Yellow cadaverine biotin-X. The biotinylated Lucifer Yellow is visualized by incubation of the slice overnight in avidin-biotin-HRP complex in the presence of 0.1% Triton X-100 at 4 degrees C. A diaminobenzidine reaction with simultaneous cobalt and nickel intensification follows. This method produces well-filled neuronal cell bodies, dendrites, and spines for light microscopic analysis. The non-fluorescent reaction product in these intracellularly filled cells may permit transmitter immunohistochemistry and synaptic fine structure to be studied in combination with neural connections and dendritic morphology.
本文描述了一种新方法，该方法可通过逆行运输、细胞内注射生物素标记的路西法黄以及生物素的组织化学反应来识别神经元。通过将罗丹明标记的乳胶珠注射到内侧膝状体并在体内进行逆行运输，可识别出投射到同侧丘脑的下丘中的细胞。几天后，用醛类固定大脑，并在振动切片机上切割下丘。用落射荧光光学系统观察罗丹明标记的细胞，在视觉控制下用细胞内微电极刺入细胞，并注射9%的路西法黄二锂盐和1%的路西法黄尸胺生物素-X。在4℃下，将切片在含有0.1% Triton X-100的抗生物素蛋白-生物素-辣根过氧化物酶复合物中孵育过夜，使生物素化的路西法黄可视化。随后进行二氨基联苯胺反应，并同时增强钴和镍。该方法可产生填充良好的神经元细胞体、树突和棘突，用于光学显微镜分析。这些细胞内填充细胞中的非荧光反应产物可能允许结合神经连接和树突形态学来研究递质免疫组织化学和突触精细结构。
