Cobalt uptake and localization of putative calcium binding sites in striated muscle

Frog sartorius muscles were soaked overnight in saline containing 50 mM cobalt and subsequently fixed with or without precipitation of intracellular cobalt by exposure to ammonium sulfide. Treated fibers and appropriate controls were embedded in plastic in the usual manner. Thick sections (3-4 micrometers) of each sample were treated with silver in order to intensify precipitated cobalt. The samples were then resectioned for ultrastructural examination. Fibers exposed to cobalt with or without precipitation had normal ultrastructure; indeed, the transverse lines of the A-zone and M-band appeared to be more obvious after exposure to cobalt than in control samples. Exposure to silver produced numerous silver grains only in those samples that contained precipitated cobalt. The distribution of silver grains after intensification matched expected calcium binding sites. Silver grains were abundant over membranes of the sarcoplasmic reticulum, sarcolemma, mitochondria, and nucleus and rare or absent from filaments and sarcoplasm. This technique appears to be generally useful for locating intracellular calcium binding sites.