Revisiting sodium phosphotungstate and ammonium molybdate as nonradioactive negative-staining agents for single-particle analysis.

Abstract

This study reports the successful replacement of uranyl-based stains by either sodium phosphotungstate or ammonium molybdate in negative-staining electron microscopy. Using apoferritin as a test specimen, it is demonstrated that in combination with a facile on-grid fixation step, both stains yield comparable images to uranyl formate. Subsequently, using β-galactosidase, it is shown that both stains can also successfully be employed for single-particle analysis, yielding virtually indistinguishable results from uranyl formate. As both replacement stains are nonradioactive, they are not subjected to the same handling restrictions as uranyl-based stains. Therefore they are not only cheaper to use, but also make decentralized sample-grid preparation, directly after purification, accessible to a broader range of scientists.