PHOTOCONVERSION OF DIAMINOBENZIDINE WITH DIFFERENT FLUORESCENT NEURONAL MARKERS INTO A LIGHT AND ELECTRON-MICROSCOPIC DENSE REACTION-PRODUCT
PHOTOCONVERSION OF DIAMINOBENZIDINE WITH DIFFERENT FLUORESCENT NEURONAL MARKERS INTO A LIGHT AND ELECTRON-MICROSCOPIC DENSE REACTION-PRODUCT
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DOI：
10.1002/jemt.1070240103
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发表时间：
1993-01-01
影响因子：
2.5
通讯作者：
LUBKE, J
中科院分区：
文献类型：
--
作者：
LUBKE, J
本文介绍了在含有荧光染料的神经元中将二氨基联苯胺（DAB）光转化为稳定的、光和电子显微镜下可见的暗反应产物的方法。到目前为止，DAB已通过以下荧光染料实现了光转化：罗丹明标记乳胶微球（RLM）， 4,6-二氨基-2-苯基吲哚（DAPI）， 5,7-二羟色胺（5,7- dht）， Fast Blue (FB)，核黄（NY），二氨基黄（DY）， Evans Blue (EB)，吖啶橙（AO），溴化乙啶（EBR），1,1'-二十二烷基-3,3,3',3'-四甲基吲哚高氯酸盐，D-282 (DiI)，碘化丙啶（PI）和细胞内注射的路西法黄（LY）。染料通过着色染色、逆行运输或细胞内注射进入神经元。光转化是通过将组织与含有荧光物质的细胞在DAB溶液中同时在紫外线（UV）强光照射下孵育进行的。在反应产物形成过程中，荧光从细胞中消失。在所有情况下，光转换为光学显微镜提供了稳定，不褪色的DAB反应产物。此外，在电子显微镜水平上，似乎光转化导致反应产物分布均匀，颗粒细，颜色深，位于细胞内。在大多数逆行示踪剂测试中，光转化只导致细胞体和初级树突近端部分的染色。在细胞内充满ly的神经元和逆行标记DiI、DiO和5,7- dht的细胞进行光转化后，反应产物存在于整个细胞中，从细胞体延伸到树突和树突阑尾，并延伸到轴突。荧光染料将DAB光转化为稳定的、光镜下和电子显微镜下的致密反应产物，具有高选择性和方法简单的特点，为传统的神经解剖技术提供了一个有希望的替代方案，也为荧光神经元示踪剂在光镜和电子显微镜下的新应用提供了有益的途径。
This article describes methods for photoconverting diaminobenzidine (DAB) into a stable, light and electron microscopically visible dark reaction product in neurons which contain a fluorescent dye. Photoconversion of DAB has been achieved so far with the following fluorescent dyes: rhodamine labeled latex microspheres (RLM), 4,6-diamidino-2-phenylindole (DAPI), 5,7-dihydroxytryptamine (5,7-DHT), Fast Blue (FB), Nuclear Yellow (NY), Diamidino Yellow (DY), Evans Blue (EB), acridine orange (AO), ethidium bromide (EBR),1,1'-dioctadecyl-3,3,3',3'-tetramethylindolcarbocyanine perchlorate, D-282 (DiI), propidium iodide (PI), and intracellularly injected Lucifer Yellow (LY). The dye is introduced into the neurons by tinctorial staining, retrograde transport, or intracellular injection. Photoconversion is conducted by incubating the tissue with the fluorescent substance-containing cells in a DAB solution under simultaneous strong illumination with ultraviolet (UV) light. During the formation of the reaction product, the fluorescence disappears from the cell. In all cases, photoconversion provided a stable, nonfading DAB reaction product for light microscopy. In addition, at the electron microscopic level, it appeared that the photoconversion results in a homogeneously distributed, fine granular, dark, intracellularly located reaction product. With most of the retrograde tracers tested, photoconversion led only to staining of the cell bodies and the proximal portions of primary dendrites. Following photoconversion with intracellularly LY-filled neurons and cells labeled retrogradely with DiI, DiO, and 5,7-DHT, the reaction product was present throughout the cells, extending from the cell bodies into dendrites and dendritic appendices, and into axons. The high selectivity and methodological simplicity of photoconversion of DAB with fluorescent dyes into a stable, light and electron microscopical dense reaction product provide a promising alternative to classical neuroanatomical techniques and a new useful application of fluorescent neuronal tracers to light and electron microscopy.