Castellano B, González B, Jensen M B, Pedersen E B, Finsen B R, Zimmer J
Department of Cell Biology and Physiology, Autonomous University of Barcelona, Spain.
J Histochem Cytochem. 1991 May;39(5):561-8. doi: 10.1177/39.5.1707903.
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.
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。该程序包括一种针对小胶质细胞的组织化学染色和一种针对星形胶质细胞的免疫细胞化学染色，将染色产物的最终显色推迟到两个反应都完成之后。首先，通过核苷二磷酸酶（NDPase）的组织化学反应对小胶质细胞进行特异性但不可见的标记。然后，通过进行胶质纤维酸性蛋白（GFAP）免疫细胞化学反应的第一部分来标记星形胶质细胞。最后，在一系列中间步骤中，通过用硫化铵和硝酸银处理使NDPase反应产物显色并稳定，同时使用1-萘酚碱性染料法使GFAP免疫反应产物显色。最终产物中，NDPase阳性的小胶质细胞呈棕色，GFAP反应性的星形胶质细胞呈蓝色。在大鼠脑组织的振动切片和原代星形胶质细胞培养物中，这两种类型的胶质细胞清晰可辨，并且我们从未观察到同时对NDPase和GFAP染色的细胞。当用识别小胶质细胞和巨噬细胞的OX-42抗体替代GFAP抗体时，观察到了小胶质细胞的双重染色。该染色方案在正常脑以及神经元或轴突变性的不同病理状态下小胶质细胞与星形胶质细胞之间功能相互作用的研究中具有广泛应用，同样也可用于细胞培养的实验研究。
