Specificity of the zinc-iodide-osmium (ZIO) reagent in the nervous tissue: evaluation of the Ca++ binding sites hypothesis.

We tested the Ca(2+) binding sites detection hypothesis of the zinc-iodide-osmium (ZIO) staining on synaptic vesicles of rat cerebellar glomerulus using three different approaches based on the previous observation that Ca(2+) chelator EGTA can impede the staining reaction: (a) ZIO staining of "en bloc" tissue samples after EGTA exposure, (b) staining of ultrathin sections with ZIO reagent after EGTA treatment, (c) ZIO staining of isolated synaptic vesicles, preincubated in calcium ionophore A23187. The results of our investigation do not support the hypothesis. We conclude that ZIO staining can be referred to different molecules located mainly in secretory organelles.