Negative staining and cryo-negative staining: Applications in biology and medicine

Abstract

Negative staining is widely applicable to isolated viruses, protein molecules, macromolecular assemblies and fibrils, subcellular membrane fractions, liposomes and artifi cial membranes, synthetic DNA arrays, and also to polymer solutions and a variety of nanotechnology samples. 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 benefi t 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 floating and carbon sandwich techniques in addition to the negative staining-carbon film (NS-CF) technique for randomly dispersed fragile molecules, 2D crystallization of proteins and for cleavage of cells and organelles. Immuno-negative staining and negative staining of affi nity labeled complexes (e.g., biotin-streptavidin) are presented in some detail. The formation of immune complexes in solution for droplet negative staining is given, as is the use of carbon- plastic support films as an adsorption surface on which to perform immunolabeling or affi nity experiments, prior to 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. The more recently developed cryonegative staining procedures are also included: first, the high concentration ammonium molybdate procedure on holey carbon films and second, the carbon sandwich procedure using uranyl formate. Several electron micrographs showing examples of applications of negative staining techniques are included and the chapter is thoroughly referenced. © Springer Science+Business Media New York 2014.