A DOUBLE STAINING TECHNIQUE FOR SIMULTANEOUS DEMONSTRATION OF ASTROCYTES AND MICROGLIA IN BRAIN SECTIONS AND ASTROGLIAL CELL-CULTURES
A DOUBLE STAINING TECHNIQUE FOR SIMULTANEOUS DEMONSTRATION OF ASTROCYTES AND MICROGLIA IN BRAIN SECTIONS AND ASTROGLIAL CELL-CULTURES
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DOI：
10.1177/39.5.1707903
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发表时间：
1991-05-01
影响因子：
3.2
通讯作者：
ZIMMER, J
中科院分区：
文献类型：
--
作者：
CASTELLANO, B;GONZALEZ, B;ZIMMER, J
我们开发了一种双重染色技术，用于在组织学脑切片和细胞培养物中同时显示星形胶质细胞和小胶质细胞。 该程序包括对小胶质细胞特异性的组织化学染色和对星形胶质细胞特异性的免疫细胞化学染色，并推迟染色产物的最终可视化，直到进行了两种反应。 首先，小胶质细胞特异性，但不可见的核苷二磷酸酶（NDP 3）的组织化学反应标记。 然后通过进行胶质细胞酸性蛋白（GFAP）免疫细胞化学反应的第一部分标记星形胶质细胞。 最后，在一系列的干预步骤中，通过用硫化铵和硝酸银处理使NDV反应产物可视化并稳定化，而1-萘酚碱性染料法用于使GFAP免疫反应产物可视化。 作为最终产物，NDPase阳性小胶质细胞为棕色，GFAP反应性星形胶质细胞为蓝色。 这两种类型的神经胶质细胞在大鼠脑组织的振动切片和原代星形胶质细胞培养物中可以清楚地区分，并且我们从未观察到NDT和GFAP染色的细胞。 当GFAP抗体被识别小胶质细胞和巨噬细胞的OX-42抗体取代时，观察到小胶质细胞的双重染色。 染色协议有广泛的应用，在正常的大脑和不同的病理状态与神经元或轴突变性的小胶质细胞和星形胶质细胞之间的功能相互作用的研究，就像它可以用于细胞培养的实验研究。
We developed a double staining technique for simultaneous demonstration of astrocytes and microglial cells in histological brain sections and cell cultures. The procedure included a histochemical stain specific for microglial cells and an immunocytochemical stain specific for astroglial cells, with postponement of the final visualization of the staining products until both reactions had been performed. First, microglial cells were specifically but invisibly labeled by histochemical reaction for nucleoside diphosphatase (NDPase). Then the astroglial cells were labeled by performing the first parts of the immunocytochemical reaction for glial fibrillary acidic protein (GFAP). Finally, in a series of intervening steps, the NDPase reaction product was visualized and stabilized by treatment with ammonium sulfide and silver nitrate, while the 1-naphthol basic dye method was used to visualize the GFAP immunoreactive product. As an end product, the NDPase-positive microglial cells were brown and the GFAP-reactive astroglial cells blue. The two types of glial cells were clearly distinguishable in vibratome sections of rat brain tissue and in primary astroglial cell cultures, and we never observed cells that stained for both NDPase and GFAP. When the GFAP antibody was replaced by the OX-42 antibody, which recognizes microglial cells and macrophages, double staining of microglial cells was observed. The staining protocol has wide applications in studies of the functional interactions between microglial and astroglial cells in the normal brain and in different pathological states with neuronal or axonal degeneration, just as it can be used for experimental studies in cell cultures.