Immersion Autometallography: Histochemical In Situ Capturing of Zinc Ions in Catalytic Zinc-Sulfur Nanocrystals

Abstract

S U M M A R YIn the mid-1980s, two versions of Timm's original immersion sulfide silver method were published. The authors used immersion of tissue in a sulfide solution as opposed to Timm, who used immersion of tissue blocks in hydrogen sulfide-bubbled alcohol. The autometallography staining resulting from the "sulfide only immersion" was not particularly impressive, but the significance of this return to an old approach became obvious when Wenzel and co-workers presented their approach in connection with introduction by the Palmiter group of zinc transporter 3 (ZnT3). The Wenzel/Palmiter pictures are the first high-resolution, high-quality pictures taken from tissues in which free and loosely bound zinc ions have been captured in zinc-sulfur nanocrystals by immersion. The trick was to place formalin-fixed blocks of mouse brains in a solution containing 3% glutaraldehyde and 0.1% sodium sulfide, ingredients used for transcardial perfusion in the zinc-specific NeoTimm method. That the NeoTimm technique results in silver enhancement of zinc-sulfur nanocrystals has been proved by proton-induced X-ray multielement analyses (PIXE) and in vivo chelation with diethyldithiocarbamate (DEDTC). The aims of the present study were (a) to make the immersion-based capturing of zinc ions in zinc-sulfur nanocrystals work directly on sections and slices of fixed brain tissue, (b) to work out protocols that ensure zinc specificity and optimal quality of the staining, (c) to apply "immersion autometallography" (iZnS AMG ) to other tissues that contain zinc-enriched (ZEN) cells, and (d) to make the immersion approach work on unfixed fresh tissue.

Mobile zinc increases rapidly in the retina after optic nerve injury and regulates ganglion cell survival and optic nerve regeneration

“…These chelatable Zn 2+ ions, which in neurons are localized primarily in synaptic vesicles of so-called zinc-enriched neurons, can be detected by either fluorescence or AMG techniques. Although AMG detects copper (85) and iron (86), as well as zinc (62,87,88), ZP1 is highly selective for Zn 2+ and its intrinsic fluorescence is quenched by copper, iron, manganese, and cobalt ions (38,66,89). Thus, the fact that we observed similar changes in Zn 2+ with AMG and ZP1 reinforces the conclusion that elevation of Zn 2+ itself is occurring after NC.…”

Zinc-specific Autometallographic In Vivo Selenium Methods: Tracing of Zinc-enriched (ZEN) Terminals, ZEN Pathways, and Pools of Zinc Ions in a Multitude of Other ZEN Cells

“…Another valuable quality of the technique is that zinc-selenium nanocrystals are more resistant to weak acids than are the zinc-sulfur nanocrystals, the silver-enhanceable entities of the NeoTimm methods (Danscher 1981(Danscher ,1996Stoltenberg and Danscher 2000;Danscher et al 2004). This relative pH stability of the zinc-selenium nanocrystals is also the quality that makes it possible to trace the neuronal somata of ZEN neurons.…”

Topographical Analysis of Reactive Zinc in the Central Nervous System of Adult Zebrafish (Danio Rerio)

“…31 Briefly, each zebrafish CNS was immersed in 2 mL of 3% glutaraldehyde solution in a 0.1 M phosphate buffer (pH 7.4) for 24 h, and then moved to 2 mL of a sodium sulfide solution (1% Na 2 S) in a 0.12 M Millonig's buffer (97 mM NaOH, 138 mM NaH 2 PO 4 , and 0.18 mM CaCl 2 ) for 24 h. Later, each sample was embedded in 4% agarose (Invitrogen) and glued with cyanoacrylic adhesive to the stage of a vibroslicer (VTS-1000; Leica). Coronal (30-lm-thick) slices were cut in phosphatebuffered saline, mounted on gelatinized slides, and left to dry.…”