Harris J Robin
Instiute of Zoology, University of Mainz, Mainz, Germany.
Methods Mol Biol. 2007;369:107-42. doi: 10.1007/978-1-59745-294-6_7.
Negative staining is widely applicable to isolated viruses, protein molecules, macro-molecular assemblies and fibrils, subcellular membrane fractions, liposomes and artificial membranes, synthetic DNA arrays, and also to polymer solutions. In this chapter, techniques are provided for the preparation of the necessary support films (continuous carbon and holey/perforated carbon). The range of suitable negative stains is presented, with some emphasis on the benefit of using ammonium molybdate and of negative stain-trehalose combinations. Protocols are provided for the single-droplet negative staining technique (on continuous and holey carbon support films), the negative staining-carbon film technique, for randomly dispersed fragile molecules, 2D crystallization of proteins, and for cleavage of cells and organelles. The newly developed cryonegative staining procedure also is included. Immunonegative staining and negative staining of affinity labeled complexes (e.g., biotin-streptavidin) are discussed in some detail. The formation of immune complexes in solution for droplet negative staining is presented, as is the use of carbon-plastic support films as an adsorption surface on which to perform immunolabeling or affinity experiments, before negative staining. Dynamic biological systems can be investigated by negative staining, where the time period is in excess of a few minutes, but there are possibilities to greatly reduce the time by rapid stabilization of molecular systems with uranyl acetate or tannic acid.
负染色广泛应用于分离的病毒、蛋白质分子、大分子聚集体和纤维、亚细胞膜组分、脂质体和人工膜、合成DNA阵列，以及聚合物溶液。在本章中，将介绍制备必要支撑膜（连续碳膜和多孔/穿孔碳膜）的技术。列出了合适的负染色剂范围，重点介绍了使用钼酸铵和负染色剂-海藻糖组合的优点。提供了单滴负染色技术（在连续和多孔碳支撑膜上）、负染色-碳膜技术、随机分散的易碎分子、蛋白质二维结晶以及细胞和细胞器裂解的实验方案。还包括新开发的低温负染色程序。详细讨论了免疫负染色和亲和标记复合物（如生物素-链霉亲和素）的负染色。介绍了用于液滴负染色的溶液中免疫复合物的形成，以及在负染色前使用碳-塑料支撑膜作为吸附表面进行免疫标记或亲和实验的方法。负染色可用于研究动态生物系统，其时间周期超过几分钟，但通过用醋酸铀或单宁酸快速稳定分子系统，有可能大大缩短时间。
