The autometallographic zinc-sulphide method. A new approach involving in vivo creation of nanometer-sized zinc sulphide crystal lattices in zinc-enriched synaptic and secretory vesicles.

A new version of Timm's sulphide silver method involving in vivo binding of zinc ions in zinc enriched terminals is presented. By injecting sodium sulphide into the vena cava of deeply anaesthetized animals, it is possible to bind chemically the vesicular zinc, i.e. chelatable zinc (zinc ions), in secretory and synaptic vesicles, in the form of zinc sulphide crystal lattices. Four minutes after the intravenous injection the animal is perfused transcardially with a phosphate-buffered solution of glutaraldehyde, glutaraldehyde and formaldehyde, or with a saline solution. The nanometer-sized catalytic crystals can then be silver-amplified in cryostat and vibratome sections by exposure to an autometallographic developer. It is demonstrated that contemporaneously with silver enhancement, the zinc sulphide crystals are transformed to the corresponding silver sulphide crystals. For ultrastructural studies, autometallographic development of vibratome sections is recommended. From these sections tissue blocks are cut from the areas of interest, blockstained with osmium tetroxide and embedded in Epon. This approach results in a zinc-specific autometallographic staining of the sections of a hitherto unseen, high technical quality.