A double staining technique for simultaneous demonstration of astrocytes and microglia in brain sections and astroglial cell cultures.

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.