Immersion Autometallography : Histochemical In Situ Capturing of Zinc Ions in Catalytic ZincSulfur Nanocrystals
Department of Neurobiology, Institute of Anatomy, University of Aarhus, Aarhus, Denmark
Correspondence to: Prof. Gorm Danscher, Dept. of Neurobiology, Inst. of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark. E-mail: gd{at}neuro.au.dk
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Summary |
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Key Words: immersion autometallography (iZnSAMG) zinc ions brain zinc-enriched (ZEN) ZEN terminals NeoTimm
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Introduction |
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Physical development was used by photographers for artistic purposes to manipulate photos. The light-exposed photographic plate was depleted of its silver bromide crystals by thiosulfate, leaving only the tiny traces of metallic silver grains resulting from the light exposure. The invisible picture was then made visible by being developed in a "physical developer," a misleading name based on the misconception that the silver amplification performed under these circumstances was different from the normal "chemical development" (Danscher 1996).
Since Timm's introduction of the concept that some metal sulfide accumulations could be silver-enhanced by physical development (Timm 1958,1962
) many scientists have used his approach, have tried to improve the technique, and have added new metals to the family of nanocrystals that can be silver-enhanced (e.g., Haug 1967
,1973
,1974
; Brunk and Brun 1972
; Danscher 1981a
,b
; Sloviter 1990
; Danscher and Møller-Madsen 1985
; Zdolsek et al. 1993
; Ross et al. 1996
). In the early 1980s, Timm's original sulfide silver method was made zinc-specific (Danscher 1981c
), and the selenium technique for in vivo creation of zincselenium nanocrystals in zinc-enriched (ZEN) neuronal terminals and for retrograde axonal tracing of ZEN neurons was introduced (Danscher 1982
,1984a
; Howell and Frederickson 1990
). As a result of the growing understanding of the processes underlying silver enhancement, the term autometallography (AMG) was suggested to replace the term "physical development" when used in histochemistry (Danscher 1984b
). This was particularly relevant because it was proved that metallic gold nanoparticles were the substrate for AMG silver enhancement in tissues from animals or humans that had been subjected to gold salts (Danscher 1981b
). This finding led to the use of AMG for silver-enhancement of colloidal gold particles in immunohistochemistry (IHC) (Holgate et al. 1983
) and for histochemical tracing of enzymes (Danscher and Nørgaard 1983
).
Cassell and Brown (1984) and Tauck and Nadler (1985)
described a version of Timm's sulfide silver method in which immersion in alcohol bubbled with hydrogen sulfide was substituted by the buffered sodium sulfide solution introduced by Haug for transcardial perfusion (for details see Haug 1973
). This solution was also used in a 1% version for transcardial perfusion by Danscher and Zimmer (1978)
and was finally corrected to 0.1% in the zinc ion-specific version of Timm's method, the so-called NeoTimm method (Danscher 1981c
).
The 0.1% solution of buffered sodium sulfide was used to immerse human brain blocks by Franck et al. (1995) and Sutula et al. (1989)
. However, not until the Wenzel/Palmiter/Cole pictures from mouse brains, immersed in the combined glutaraldehydesodium sulfide solutions of the NeoTimm technique, did the benefit of this approach become appreciable (Wenzel et al. 1997
; Cole et al. 1999
; Franco-Pons et al. 2000
).
The present study aims at establishing correctly performed immersion autometallography (iZnSAMG) as a highly specific tool for tracing zinc ions directly in tissue sections and to give easy-to-follow protocols of the technique and ways of controlling zinc specificity.
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Materials and Methods |
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The animals were deeply anesthetized IP with Mebumal and sacrificed by decapitation or by transcardial perfusion with a fixative at a pressure of 120130 mmHg. The perfusions were initiated by prewashing with an NaClheparin solution [9 ml 0.9% NaCl + 1 ml heparin (5000 IU/ml)] for 30 sec followed by transcardial perfusion with either (a) 250 ml 4% paraformaldehyde for 15 min, or (b) 250 ml fixative containing 1% paraformaldehyde and 1% glutaraldehyde for 15 min, or (c) 250 ml fixative containing 3% glutaraldehyde for 15 min, but without prewashing. All solutions were buffered with a 0.15 M Sørensen phosphate buffer.
Based on a multitude of experiments, the following procedures were found to be superior.
Immersion Procedure
One-, 2-, 4-, 12-, 16-, and 20-mm-thick tissue slices were cut by placing the brains in the "fast tissue slicer" (Histotech; Copenhagen, Denmark). The slices were immersed in a NeoTimm solution (NTS), i.e., 0.1% sodium sulfide and 3% glutaraldehyde in a 0.1 M Sørensen phosphate buffer, pH 7.4. The immersion jars were placed on a shaker and kept at 4C. Three days later the slices were carefully rinsed twice in 0.1 M phosphate buffer for 10 min. The fixed slices were further processed with or without AMG development.
Non-developed NTS Slices
AMG-developed NTS Slices
The AMG Silver Lactate Enhancement Developer (pH 3.8)
The AMG developer consists of a 60-ml gum arabic solution and 10 ml sodium citrate buffer (25.5 g of citric acid 1 H2O + 23.5 g sodium citrate 2 H2O to 100 ml distilled water), 15 ml reductor (0.85 g of hydroquinone dissolved in 15 ml distilled water at 40C), and a 15-ml solution containing silver ions (0.12 g silver lactate in 15 ml distilled water at 40C) added immediately before use while thoroughly stirring the AMG solution (Danscher 1981a; Stoltenberg and Danscher 2000
).
AMG Development of Tissue Sections Placed on Glass Slides
The glass slides were placed in Farmer-cleaned jars, poured with the AMG developer and placed in a water bath at 26C on an electric device that shook the jars gently. The entire set-up was prepared in plain daylight on the lab bench but was covered by a dark hood throughout the actual development. After 60 min the AMG development was stopped by replacing the developer with a 5% sodium thiosulfate solution for 10 min (AMG stop bath). The jars were then placed under gently running ion-exchange water for 5 min.
If the tissue sections reveal a high background staining, two approaches have been worked out: (a) The glass slides are dipped in a 0.5% solution of gelatin and allowed to dry before development. After AMG the jars are placed under 45C warm tapwater for 15 min before they are rinsed in distilled water, counterstained, and coverslipped. (b) The glass slides are dipped in Farmer solution for 1030 sec, rinsed in distilled water, counterstained, and coverslipped (for details see Danscher 1996).
AMG Development of NTS Slices
The slices were placed in Farmer-cleaned jars and poured with the AMG developer. The jars were placed in a water bath at 26C and covered by a light-tight hood. After 60 min the development was stopped by replacing the developer with 5% sodium thiosulfate. After 10 min the slices were rinsed carefully for 5 min in several washes of distilled water.
Human Tissue
Human tissues obtained from autopsies or biopsies were fixed in 4% buffered formalin for 2 hr before being immersed in the NTS for 3 days. Sections for LM and EM analysis were performed as described above. If the tissue blocks are less than 2 mm in diameter, they can be placed in the NTS without previous cutting.
Alternative Ways of Performing iZnSAMG
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Results |
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Electron Microscopy
The excellent quality of the technique at the ultrastructural level is demonstrated in Figure 2.
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Human Brain
Figure 1F demonstrates ZEN terminals in neocortex from a brain autopsy. The procedures are summarized in Figure 3.
iZnSAMG Staining of Zinc Ions in Zinc-enriched Cells
The iZnSAMG technique works well on other types of ZEN cells, including pancreas (Figure 1E), gut, prostate, liver, and kidney.
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Discussion |
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The approach is easy to control for specificity by chelation, e.g., by treating live animals or the cultures/cell samples with diethyldithiocarbamate (DEDTC), whereby zinc ions are bound in vivo as zinc DEDTC, or by placing the tissue slices directly into solutions of DEDTC or TSQ before the NTS immersion.
The possibility of creating the more stable zincselenium nanocrystals by immersion in a solution of sodium selenide and glutaraldehyde by using the more stable selenide ions instead of sulfide ions, i.e., creating zincselenium (ZnSe) nanocrystals, has been tested and found not to be an option. We do not know why sodium selenide creates zincselenium nanocrystals when applied in vivo or by transcardial perfusion (Danscher 1982,1984b
,c
,d
; Slomianka et al. 1990
), but is of no value for immersion capturing of zinc ions.
We recommend the following AMG approaches for specific histochemical tracing of zinc ions in neuronal terminals: (a) transcardial perfusion with 0.1% sodium sulfide and phosphate-buffered 3% glutaraldehyde (the NeoTimm method); (b) immersion of up to 2-mm slices of tissue in the NeoTimm solution (immersion autometallography); (c) in vivo treatment with sodium selenite systemically, i.e., by IV or IP application, or locally with sodium selenide, e.g., by intracerebral injection, and survival times from 1 to 2 hr for tracing ZEN terminals in CNS and PNS; (d) systemic or local injection of, respectively, sodium selenite and selenide and survival times from 18 to 36 hr for tracing ZEN fiber tracts by retrograde axonal loading of ZEN somata.
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Acknowledgments |
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We gratefully acknowledge the skillful technical assistance of Ms Helene Andersen, Mr A. Meier, Ms H. Mikkelsen, Ms Lene Munkøe, Mr T.A. Nielsen, Ms M. Sand, and Ms K. Wiedemann.
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Footnotes |
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Literature Cited |
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