The use of electron microscopic immunocytochemistry with silver-enhanced 1.4-nm Gold particles to localize GAD in the cerebellar nuclei

Abstract

Silver enhancement of small gold particles can be used with pre-embedding immunocytochemistry to analyze the distribution of label over cell organdies. We have developed a method that improves tissue morphology, has good penetration of reagents, and allows greater control of silver enhancement of 1.4-nm gold. In this study we analyzed the distribution of glutamic acid decarboxylase (GAD), a synthetic enzyme for the inhibitory neurotransmitter γ-aminobutyric acid (GABA), in the cerebellar nuclei of the mouse. Pre- embedding immunocytochemistry was carried out on brain sections fixed with high concentrations of glutaraldehyde and sodium metabisulfite. After incubations with a monoclonal antibody to GAD and a 1.4-nm NanoGold-labeled secondary antibody, sections were silver-enhanced with N-propyl gallate as a reducing agent and MES as a new buffer system. In the cerebellar nuclei, GAD label was specifically localized in axon terminals over dusters of synaptic vesicles. These terminals formed axosomatic and axodendritic contacts. The majority of GAD-labeled terminals had cytological characteristics indicating their origin from Purkinje cells, which are known to contain GAD.