Localization of cations by pyroantimonate. II. Electron probe microanalysis of calcium and sodium in skeletal muscle of mouse.

A new formulation of the pyroantimonate (PA) method for localization of calcium and sodium is proposed and evaluated in mouse skeletal muscle. This study, performed at the ultrastructural level by means of transmission electron microscopy (TEM) and electron probe microanalysis (EPMA), completes a previous work done at the optical level with analytical ion microscopy (AIM), which enabled us to define the appropriate composition of fixatives. In our present experiments, calcium and sodium were shown localized in various cell structures, e.g., T-tubules, glycogen, granules, nuclei. For AIM, the best fixatives were characterized by PA supersaturation, which resulted in smaller crystals and a high rate of penetration in the presence of paraformaldehyde and either phenol or collidine. Contrary to the findings at the optical level, collidine did not give satisfactory results at the ultrastructural level. The method of floating sections on the microtome trough was an important cause of cation displacement. We found that alkalinization of the floating medium significantly decreased ion loss. The technique also provided an indication of the form of these elements: free or easily liberated cations were precipitated into coarse PA deposits; electron-positive chelates were "stained" by PA; neutral chelates were not stained, but some of them could be detected by EPMA. This PA method should make possible more precise localization of cellular calcium, such as in glycogen metabolism, and perhaps detection of movements of cytoplasmic calcium and sodium.