AUTOMETALLOGRAPHIC SILVER ENHANCEMENT OF ZINC-SULFIDE CRYSTALS CREATED IN CRYOSTAT SECTIONS FROM HUMAN BRAIN BIOPSIES - A NEW TECHNIQUE THAT MAKES IT FEASIBLE TO DEMONSTRATE ZINC IONS IN TISSUE-SECTIONS FROM BIOPSIES AND EARLY AUTOPSY MATERIAL

We present a new technique that allows zinc ions in synaptic and secre tory vesicles of biopsy and early autopsy material (<2 hr post mortem) to be transformed to nanometer-sized zinc sulfide crystal lattices fo r subsequent autometallographic (AMG) development. Human brain biopsie s, or other tissue samples containing zinc-enriched (ZEN) cells, are f rozen in liquid nitrogen or by CO2 gas immediately after removal. The tissue blocks are cut in a cryostat and the sections placed on glass s lides. The slides are transferred to an H2S exposure chamber placed in a -15C freezer. After 1-24 hr of gas exposure the sections are remove d from the chamber, fixed while thawing, and dehydrated. The sections are then exposed to an AMG developer. AMG causes silver enhancement of zinc sulfide crystal lattices created in the tissues through the H2S exposure, making them visible. It is imperative that the tissues are f rozen instantaneously after removal, because loosely bound or free zin c ions start leaving their vesicular compartment soon after death. The AMG technique can, despite inadequate fixation and damage to the tiss ue caused by freezing, also be used to trace zinc ions at ultrastructu ral levels, and it is demonstrated that zinc ions in the human neocort ex are located in synaptic vesicles. In the few human biopsies analyze d thus far, the light microscopic pattern created by the silver-enhanc ed ZEN terminals resembles that seen in the neocortex of rat brain. Th e technique has been applied to cryostat sections from neocortex biops ies of five individuals undergoing brain surgery. Biopsies from three patients resulted in satisfactory AMG-stained sections. Rat brains rem oved and frozen immediately after decapitation constituted the materia l on which the present technique was developed. Such material results in an almost uniform high quality of staining, and we found that unexp osed sections can be stored for at least 5 months at -80C without ensu ing significant loss of AMG staining intensity.