A freeze-substitution method for localizing divalent cations: examples from secretory systems

This paper presents a simple method for localizing calcium in its natural distribution. This method depends on freeze-substituting rapid-frozen tissue, and combines the spatial precision of conventional plastic sections with the reliable calcium localization characteristic of direct freezing methods. Calcium is retained in the tissue during freeze-substitution and subsequent processing, and its distribution in frog skeletal muscle suggests that it is not displaced during tissue processing or in the electron probe. Examples of new findings with this method are the presence of a postsynaptic sequestration system for cations entering at the neuromuscular junction, and a nonmitochondrial system in nerve muscle synapses that sequesters cations entering during depolarization-secretion coupling.