Significance of the maillet method (ZIO) for cytochemical studies of subcellular structures

The present knowledge on the methodology, specificity, localization, cytochemical significance and experimental variations of the zinc iodide-osmium tetroxide (ZIO) staining are reviewed. Some new findings supporting the concept that ZIO reacts with -SH groups in some structures under specific conditions are also communicated. In this respect the effect of in vitro pretreatment with -SH reagents on ZIO staining, already described in different kinds of synaptic vesicles, was studied in rod photoreceptor outer segments. It was observed that the intra- and extradiskal electron opaque deposits, present in untreated rod outer segments directly stained with ZIO at 4 degrees C for 2 h, were enhanced by previous incubation with dithioerythrytol (DTE), which protects -SH groups and reduce -S-S-bridges; when N-ethyl-maleimide (NEM), a -SH blocking agent was used directly or after DTE, the electron opaque deposits were considerably reduced or abolished. Furthermore the influence of previous incubation with cysteine on ZIO staining was studied in the rat pineal gland. In the controls ZIO made at 4 degrees C for 2 h, reacts strongly with the matrix of the small granulated synaptic vesicles in the pineal nerves; no electron opaque deposits are seen in the membranes of the vacuolar system. Previous incubation with cysteine 5 mM in phosphate buffer makes negative ZIO reaction in the matrix of the synaptic vesicles while with a concentration of 50 mM cysteine, a partial recovery is observed. In both concentrations the membranes of the vacuolar system appeared covered by patchy electron opaque deposits.