PROGRESS IN HISTOCHEMISTRY AND CYTOCHEMISTRY Progress in Histochemistry and Cytochemistry 41 (2006) 57–139 AUTOMETALLOGRAPHY (AMG) Silver enhancement of quantum dots resulting from (1) metabolism of toxic metals in animals and humans, (2) in vivo, in vitro and immersion created zinc–sulphur/ zinc–selenium nanocrystals, (3) metal ions liberated from metal implants and particles Gorm Danscher , Meredin Stoltenberg Department of Neurobiology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark In memory of Professor Dr. Friedrich Timm Abstract Autometallographic (AMG) silver enhancement is a potent histochemical tool for tracing a variety of metal containing nanocrystals, e.g. pure gold and silver nanoclusters and quantum dots of silver, mercury, bismuth or zinc, with sulphur and/or selenium. These nanocrystals can be created in many different ways, e.g. (1) by manufacturing colloidal gold or silver particles, (2) by treating an organism in vivo with sulphide or selenide ions, (3) as the result of a metabolic decomposition of bismuth-, mercury- or silver-containing macromolecules in cell organelles, or (4) as the end product of histochemical processing of ARTICLE IN PRESS www.elsevier.de/proghi 0079-6336/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.proghi.2006.06.001 Abbreviations: AMG, autometallography/autometallographic; CCG, cationic colloidal gold; CNS, central nervous system; DEDTC, diethyldithiocarbamate; DZ, dithizone; EELS, electron energy loss spectroscopy; ELISA, enzyme-linked immunoadsorbent assays; EM, electron microscopy; EPMA, electron probe X-ray microanalysis; GA, glutaraldehyde; GGS, gold gelatin solution; IGS, immunogold staining; ip, intraperitoneal; iZnS AMG , immersion autometallographic; LM, light microscopy; MTM, membrane translocating molecules; NTS, NeoTimm solution; ntZnS AMG , NeoTimm; PAP, peroxidase anti-peroxidase; PIXE, proton-induced X-ray emission; PNS, peripheral nervous system; QD, quantum dots; ZEN, zinc-enriched; ZnSe AMG , in vivo selenium; ZnT, zinc transporter; AAS, atomic absorption spectrophotometry. Corresponding author. Tel.: +45 8942 3041; fax: +45 8618 4093. E-mail address: [email protected] (G. Danscher).
Transcript
ARTICLE IN PRESS
PROGRESS IN HISTOCHEMISTRY
AND CYTOCHEMISTRYProgress in Histochemistry and Cytochemistry 41 (2006) 57–139
0079-6336/$ -
doi:10.1016/j
Abbreviatio
central nervo
spectroscopy
electron prob
staining; ip, i
membrane tr
anti-peroxida
dots; ZEN, z
spectrophoto�CorrespoE-mail ad
www.elsevier.de/proghi
AUTOMETALLOGRAPHY (AMG)
Silver enhancement of quantum dots resulting from
(1) metabolism of toxic metals in animals and humans,
(2) in vivo, in vitro and immersion created zinc–sulphur/
zinc–selenium nanocrystals, (3) metal ions liberated from
metal implants and particles
Gorm Danscher�, Meredin Stoltenberg
Department of Neurobiology, Institute of Anatomy, University of Aarhus,
DK-8000 Aarhus C, Denmark
In memory of Professor Dr. Friedrich Timm
Abstract
Autometallographic (AMG) silver enhancement is a potent histochemical tool for tracing a
variety of metal containing nanocrystals, e.g. pure gold and silver nanoclusters and quantum
dots of silver, mercury, bismuth or zinc, with sulphur and/or selenium.
These nanocrystals can be created in many different ways, e.g. (1) by manufacturing
colloidal gold or silver particles, (2) by treating an organism in vivo with sulphide or selenide
ions, (3) as the result of a metabolic decomposition of bismuth-, mercury- or silver-containing
macromolecules in cell organelles, or (4) as the end product of histochemical processing of
see front matter r 2006 Elsevier GmbH. All rights reserved.
10.19.Quantum dot based tracing of capillaries in tissue sections . . . . . . . . . . . . 122
ARTICLE IN PRESS
Fig. 1. ‘‘Camera lucida’’ drawing of the autometallographic process. Electrons released from
hydroquinone molecules (hexagonal molecules) adhering to the gold nanocrystals (stained
gold) build up energy in the valence cloud that forms the attraction forces of the nanocrystal.
The higher energy level increases the statistical probability that silver ions (stained blue) that
have combined with the nanocrystal catch an electron and become a silver atom (stained
silver). The ‘‘new’’ silver atom shares valence electrons with the crystal valence cloud of the
original nanocrystals, i.e. has become a genuine part of the original quantum dot. As long as
the AMG development proceeds the nanocrystal will grow in size, i.e. be silver-enhanced.
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–13962
semen plasma and uncalcified bone matrix in small vesicles (Stoltenberg et al., 1997a;Danscher et al., 1999; Sørensen et al., 1999).
It has been suggested that copper, iron, aluminium and lead can be traced intissues and cell cultures by AMG after having been transformed into the respectivemetal–sulphur nanocrystal (Brunk and Brun, 1972; Wen and Wisniewski, 1985;Zdolsek et al., 1993; Domouhtsidou and Dimitriadis, 2000). If tissues are treatedwith high concentrations of sulphide ions at high pH, different kinds of metals thathave been bound in a pH dependant way to macromolecules will be released andcreate nanocrystals of metal–sulphur (Timm, 1958, 1962; Voigt, 1959; Brunk andBrun, 1972; Wen and Wisniewski, 1985; Zdolsek et al., 1993). However, lead,aluminium, and copper are not accumulated in metal–sulphur/ metal–seleniumnanocrystals as a result of metabolism, i.e. cannot be found by AMG development oftissue sections, and such nanocrystals will not be created if the exposure to sulphideions takes place at pH 7.4 or if the organism is exposed to selenium in vivo(Danscher, 1982; Danscher and Stoltenberg, 2005).
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–13964
development of the thoughts and understanding that created the indispensable webof knowledge that has made the present rather widespread use of silver amplificationin histology possible.
Zeiger, a photographic scientist, made a decisive discovery for the understandingof autometallographic catalysts when he revealed that tiny silver–sulphurnanocrystals have the same ability as metallic silver clusters to kindle the silveramplification process (Zeiger, 1938). This observation was an element in experimentsaimed at improving the light sensitivity of photographic plates/films and wasgeneralized by Timm, a specialist of forensic medicine, in his design of the silver-sulphide method (Timm, 1958). In Timm’s original method, blocks of tissue wereplaced in an alcoholic fixative bubbled with hydrogen sulphide for a period of time.In this way the tissue was fixed and exposed to sulphide ions at the same time andmetal ions, also made available by the fixation, were chemically bound to sulphideions and created metal–sulphur nanocrystals. Different kinds of sections from suchtissue blocks were then exposed to a modification of Liesegang’s developer suggestedby Luppo-Cramer (1914).
Timm knew about Roberts and Querido’s works, and this was instrumental for hiscreation of the silver-sulphide method. Another factor of guidance was the ancientknowledge that most metal sulphides are insoluble and coloured (Timm, 1961,personal communication). Interestingly, the metal that created the overwhelmingpart of the original Timm staining was zinc, which makes unstained clusters withsulphide and therefore could not have been seen in the light microscope even if thecrystals had been big enough. As a logical extension of the silver-sulphide method,Timm introduced four years later a histochemical method for tracing mercury-sulphide known to accumulate in organs of individuals and animals exposed in vivoto mercury (Timm, 1962).
Through the sixties a lot of scientists designed new versions of Timm’s silver-sulphide method (Voigt, 1959; Brunk and Brun, 1972; Sloviter, 1990). Mostimportant of these was the Timm-Haug perfusion method (Haug, 1967, 1973).Haug’s modification became the direct ancestor of the zinc specific version of Timm’soriginal method, the NeoTimm method that allowed high quality tracing of zinc ionsat LM and EM levels (Danscher, 1981a).
In 1981, it was recognized that in order to demonstrate gold in tissues from gold-exposed organisms, it was conditional that the tissue sections were exposed toreduction, either by being radiated with UV light or by being dipped in a reducingsolution; i.e., gold ions chemically bound in the tissues had to be reduced to metallicgold clusters in order to catalyze the AMG process (Danscher, 1981b). Thisknowledge was later implemented as a tool for silver enhancement of goldnanocrystals, colloidal gold particles, whether adhered to proteins like antibodiesor enzymes (Holgate et al., 1983; Danscher and Nørgaard, 1983), taken up bymacrophages (Christensen et al., 1992b), or dispersed in gelatin and infused in thevascular system (Danscher and Andreasen, 1997).
The concept of tagging macromolecules with gold was introduced by Bendayan asa tool for localizing specific antigens at ultrastructural levels (Bendayan, 1981). Thistechnique was an incredible step forward in immunohistochemistry and opened up
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139 65
possibilities of tracing the exact ultrastructural localization of a multitude ofdifferent molecules. One major problem, however, was that the new ‘‘gold-tag’’ wastoo tiny, 20–40 nm, to be studied at lower EM and LM levels. The finding that goldnanocrystals could be silver-enhanced by AMG (Danscher, 1981b) made it possibleto enhance the ‘‘gold-tag’’ signal, and the two first articles describing this approachwere published in 1983 (Holgate et al., 1983; Danscher and Nørgaard, 1983). Theintense use of immunohistochemistry in science and diagnostics quickly made thenew AMG technique quite popular, ignited industrial interests, and madecommercial gold-tagged antibodies with gold nanocrystals as small as around1 nm and AMG developers available (Hainfeld, 1987, 1988; Hainfeld and Furuya,1992; Pohl and Stierhof, 1998).
In 1981, it was also demonstrated that tissues from silver-exposed animals containnanocrystals that can be AMG developed, and it was suggested that the catalyticsilver clusters were composed of silver and sulphur (Danscher, 1981c). Proton-induced X-ray emission (PIXE) microanalysis of AMG silver grains isolated fromkidney sections of silver-exposed rats showed that the catalytic clusters weresilver–sulphur nanocrystals. This awareness of silver–sulphur and pure silvernanocrystals being AMG silver enhanceable opens up possibilities of improving awide variety of already existing histochemical techniques and inventing newhistochemical tools (Danscher, 1981a–c, 1983; Rungby et al., 1990). The AMGmyelin technique for tracing myelin in formaldehyde-fixed sections is such anexample (Larsen et al., 2003).
In the eighties it was found that certain metal–selenium nanocrystals could be tracedby AMG (Danscher, 1982, 1984a–c; Danscher andMøller-Madsen, 1985; Baatrup andDanscher, 1987). This extension in the application of AMG as a tool forneurochemical and neurotoxicological studies was based on two notable observations:(1) the detection of three elements – mercury, sulphur, and selenium – in the lysosomesof cerebellar neurons from a Minamata-diseased victim by electron microscopic X-raymicroanalysis (Shirabe, 1979), and (2) the demonstration of silver–seleniumnanocrystals in the basal lamina of the glomerular capillaries of the kidney from anargyrotic patient by crystallography (Aaseth et al., 1981). These data suggested thatmercury and silver are trapped in the organism by selenium in the same way as bysulphur and accumulate as Hg–Se or Ag–Se nanocrystals. Electron micrographs fromrat kidney sections of animals treated with mercury and selenium or silver andselenium reveal abundant AMG grains in the basement membrane of the glomerularcapillaries (Danscher, 1981c; Danscher and Møller-Madsen, 1985), and PIXE analysesof such isolated AMG grains from the different sources reveal the presence of Hg, Agand Se, respectively (Nørgaard et al., 1989, 1991).
The finding that AMG developed tissue sections, known to contain zinc-enriched(ZEN) terminals or cells, from animals treated with sodium selenite either bytranscardial perfusion or intraperitoneal (ip) injections revealed a staining patternalmost identical to that of the NeoTimm method and showed clearly that an excessamount of selenium causes the creation of zinc–selenium nanocrystals (Danscher,1982). The in vivo selenium method (ZnSeAMG) is the first technique for in vivotrapping of zinc ions and allows not only analysis of e.g. zinc ions in synaptic vesicles
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–13970
Since the human eye is relatively insensitive to the shades of AMG staining,electronic imaging can be advantageous in transforming densities to artificial colorsand can reveal otherwise undetectable dissimilarities in staining intensities (Holmet al., 1988, 1991).
Cleanliness of glassware and other tools: It is of great importance that theapparatus used to perform the AMG technique is extremely clean. The glasswareand other tools therefore have to be carefully washed in a 10% Farmer solution. It isalso imperative that high purity chemicals (pro analysis) are used in all procedures.
How to avoid unspecific background: Although cleanliness is the most importantelement in preparing high quality AMG preparations, two additional steps can betaken, both of which are described in the protocol section: (1) the sections can becovered with gelatin before AMG development; auto-developed grains in thedeveloper adhering to the surfaces of the sections can then be removed by warmwater (40 1C) after development, (2) the glass slides can be placed in a 1% Farmersolution to remove silver after development. Treatment with 1% Farmer’s solutionremoves AMG silver grains in the most superficial parts of the section, which resultsin a most satisfactory AMG staining.
Significance of light during AMG development: The acid silver lactate/gum arabicdeveloper described here is not particularly light-sensitive and development for upto 30min will cause little or no browning of the developer. Nevertheless, it isrecommended that the developing vials be wrapped in aluminium foil throughout thedevelopment, or that the water bath be covered with a light-tight box. Since thedeveloper is not that sensitive to light, the slides can be placed in the jars and coveredby the AMG developer in full daylight and then placed in the water bath through ashutter in the light-tight box.
The silver acetate developer introduced by Skutelsky et al. (1987) and Hacker et al.(1990, 1991) is an alternative to the silver lactate developer. The acetate developerhas been suggested to be less light-sensitive than the silver lactate developer in AMGdevelopers that do not contain gum arabic (Hacker et al., 1991, 1993).
AMG and osmium fixation: A particularly important technical consideration mightbe the possibility of being able to fix the AMG sections in osmium tetroxide. If highquality of tissue preservation and contrast in EM pictures are important, the tissueblocks can be stained with uranyl acetate and osmium tetraoxide after AMGdevelopment, e.g. small tissue blocks from vibratome sections developed floating inan AMG developer. It cannot, however, be recommended to gold tone the sectionsbefore osmification because the exposure to gold chloride reduces the sizes andeventually abolishes the AMG silver grains (Pohl and Stierhof, 1998).
Nickel grids and AMG: When AMG development is performed directly on thegrids, these must be made of nickel since nickel is not affected by the acid silverlactate developer. Also, they do not influence the AMG silver amplification process,and they are cheap and easy to handle.
AMG in combination with other histological techniques: AMG developed sectionsthat contain (1) colloidal gold or nanogold-tags marking antibodies or other kinds ofmacromolecules, (2) sections of tissues from animals that have been exposed to (a)sulphide ions by perfusion (NeoTimm), (b) selenide ions in vivo (the selenium
ARTICLE IN PRESS
Fig. 2. Drawing showing the two different ways autometallography can be performed. I: The
developer technique, where an AMG solution is poured into jars containing glass slides with
the tissue sections, or is dripped directly onto the individual section. II: The emulsion
technique, where the glass slides are dipped in an autoradiographic emulsion, allowed to dry
for half an hour, placed in jars and covered with a common chemical developer. Both
approaches result in the tissue sections being permeated with a solution of reducing molecules
and silver ions. As the AMG nanocrystals in the tissue sections catalyze the reduction of silver
ions to metallic silver on their surfaces either of the techniques will result in the quantum dots
becoming visible – the metal has graphed its own position in the tissue section. (Modified from
Danscher, 1991).
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139 73
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–13976
sections and have been described to give results superior to those obtained withtraditional colloidal gold probes (Hainfeld et al., 1991; Hainfeld, 1996). Undecagoldcontains eleven gold atoms only (McPartlin et al., 1969) while Nanogolds contains a1.4 nm gold nanocrystal. These gold quantum dots are bound covalently to organicgroups (Hainfeld and Furuya, 1992). Colloidal gold particles as well as Nanogolds
are used in immunohistochemistry, enzyme histochemistry, Western and dot blots,
ARTICLE IN PRESS
Fig. 6. (a) Light micrograph of AMG enhanced leukocytes filled with nanogold introduced
into the cells by way of the translocating molecule HIV-Tat peptide. The brownish tint is
caused by hundreds of thousands of AMG enhanced gold quantum dots. Bar ¼ 20mm.
(b) Electron micrograph showing that the silver-enhanced quantum dots have penetrated into
all cellular compartments. Bar ¼ 2mm.
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139 81
specific gold tagged cells in the same or other organisms by AMG development oftissue sections originating from biopsies or autopsies. The technique allows severalgenerations of cells to be traced. We have used colloidal gold particles coated with anHIV protein known as ‘‘Tat’’ (Lewin et al., 2000; Josephson et al., 1999). These‘‘nano-gold-Tat’’ units are added to isolated cells (Fig. 6) that are subsequentlyinvaded by hundreds of thousands of the units (de la Fuente and Berry, 2005;Danscher and Stoltenberg, in preparation).
3. The silver AMG techniques
Since ancient times, silver compounds have been used as a remedy for a widevariety of different illnesses; e.g., Paracelsus recommended ‘‘argentum potabile’’ as acure for epilepsy, tabes dorsalis, splenitis and hepatitis (Helling, 1967). Silver is stillwidely used in complementary and alternative medicine (CAM), where in particularcolloidal silver has been increasingly popular for the last 10 years (see e.g. http://nccam.nih.gov/health/alerts/silver/). Silver administered orally vanished frommedical practice in the beginning of the 20th century, but is still used today insilver foils to protect wounds, ointments to treat skin infections, silver nitratesolutions to swab the throat, and as the major component of amalgam fillings.Exposure to silver through mucosal membranes of the gastro-intestinal tractstill gives rise to local or generalized argyria, in particular long-termed swabbing ofthe gingiva or throat with weak silver nitrate solutions (Mehta and Dawson-Butterworth, 1966; Danscher, 1981c).
Fig. 8. (a) Cryostat section showing motor neurons loaded with retrogradely transported
silver ions in the oculomotor nucleus of rat. The silver containing protein Protagol was
injected laterally into the eye socket and the animal killed after 3 days with a transcardial
perfusion with glutaraldehyde. Bar ¼ 10 mm. (b) Darkfield micrograph from the same area
showing that the amount of silver containing neurons is high. Bar ¼ 10 mm. (c, d) Electron
micrographs showing that the retrogradely transported silver is located in lysome-like
organelles. Bars ¼ 1mm (c), 0.5mm (d).
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139 83
ARTICLE IN PRESS
Fig. 9. The silver lactate technique involves exposure of Epon embedded sections to a weak
solution of silver lactate followed by AMG enhancement of the catalytic nanocrystals that
have been created in the tissue section. The nanocrystals are primarily located in membranes,
but also other cellular structures become visible with the technique. The outcome of the
approach is so delicate that details that can otherwise be seen only at low electron
microscopical magnifications can be studied in the light microscope. (a) Section from rat
cerebellum. Bar ¼ 50mm. (b) Section from duodenum of rat. Bar ¼ 30 mm. (c) Cross section of
hair follicle in rat skin. Bar ¼ 30 mm. (d) Small area from a section of rat testis showing the
interstitium and different stages of spermatogenesis in the seminiferous tubules. Bar ¼ 30mm.
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–13986
ARTICLE IN PRESS
Fig. 10. Electron micrograph of glutaraldehyde fixed goblet cell from rat intestine. Goblet cell
mucous apparently filtering between microvilli of the adjacent absorptive cells displays an
example of seemingly selective binding of silver ions. Bar ¼ 2mm. (From Danscher, 1983).
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139 87
ARTICLE IN PRESS
Fig. 11. (a) Decalcified section from a monkey molar one year after the occlusal cavity has
been filled with amalgam. Staining is confined to the dentinal tubules. The odontoblast layer is
destroyed. Bar ¼ 50mm. (From Hørsted-Bindslev et al., 1991.) (b) Micrograph of a small part
of a lymph node taken from a rat exposed ip to silver lactate. Note the heavy load of AMG
staining in the phagocytotic cells in the lymph sinuses. Bar ¼ 30 mm. (c) Proximal tubule cell
from a rat treated with mercury chloride per os for 4 weeks and sodium selenite 0.5mg ip 6 h
before transcardial perfusion. Note precipitates in the eucromatin. m, mithochondria; n,
nucleus. Bar ¼ 1 mm. (From Danscher and Møller-Madsen, 1985). (d) Proximal tubule cell
from rat treated with mercury chloride per os for 4 weeks. Note the lack of mercury in the
nucleus compared to (c). Bar ¼ 1mm. (From Danscher and Møller-Madsen, 1985).
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–13988
ARTICLE IN PRESS
Fig. 13. (a) This micrograph from a human cryo section shows the AMG enhanced bismuth
nanocrystals in the hippocampal pyramidal neurons of CA2 and CA3. The patient had been
exposed to bismuth subnitrate for 3 months (dose unknown). AMG developed for 60min and
counterstained with toluidine blue. Bar ¼ 50mm. (b) Human neocortex from the above
patient. Bar ¼ 30mm. (c) Electron micrograph of a Helicobacter pylori culture grown for 24 h
in a bismuth-containing media, 4.6 mM of bismuth citrate. AMG enhanced bismuth
nanocrystals are seen in the bacteria. Bar ¼ 1 mm. (From Stoltenberg et al., 2001c). (d)
Electron micrograph from the rat choroid plexus. The rat was injected ip with 100mg of
bismuth subnitrate dissolved in 2ml saline and killed 1 month later. Note that bismuth is
located exclusively in lysosomes. Bar ¼ 1 mm.
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139 93
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139 95
traced with chelating molecules like dithizone (DZ) or fluorescence dyes likeZinpyr, TSQ or Zinquin in cryostat sections at low LM levels, or by autometallo-graphic silver enhancement of the same zinc ions captured in zinc–sulphur orzinc–selenium nanocrystals. The AMG techniques function at all magnifications(Figs. 14 and 15), including ultrastructural levels (Fig. 15), and the preparations canbe preserved for decades (Danscher, 1981a; Danscher et al., 2004; Danscher andStoltenberg, 2005).
Zinc ions are found in great numbers in specific vesicles, often in endo- or exocrinesecretory cells (Fig. 15d, Danscher et al., 1985a, 1999; Thorlacius-Ussing et al., 1985;Frederickson et al., 1987a; Stoltenberg et al., 1996, 1997b; Sørensen et al., 1997,1998; Kristiansen et al., 2001) or synaptic vesicles of zinc-enriched (ZEN) neurons inbrain and spinal cord (Figs. 14, 15a–c, e, f; Slomianka, 1992; Danscher et al., 1997b,2001; Rubio and Juiz, 1998; Sensi et al., 1999; Frederickson et al., 2000; Jo et al.,2000; Schrøder et al., 2000; Weiss et al., 2000; Birinyi et al., 2001; Wanget al., 2001a, b, 2002a, b; Miro-Bernie et al., 2003). They are transported intothese compartments by zinc transporters (ZnTs) that reside in the membraneof the cells and the vesicular compartments at least nine are presently known inhuman cells.
The zinc ions in some ZEN vesicles seem to be involved in packing proteins/peptides in nanocrystals (Derewenda et al., 1989; Dodson and Steiner, 1998;Søndergaard et al., 2003), but an involvement in paracrine and synaptic modulationhas also been advocated (Howell et al., 1984; Frederickson and Danscher, 1990; Kimet al., 2000; Weiss et al., 2000; Li et al., 2001; Laube, 2002; Ueno et al., 2002; Hosieet al., 2003; Ishihara et al., 2003; Cohen-Kfir et al., 2005).
The finding of zinc transporters less than a decade ago shows that zinc ionsare passing through membranes controlled by molecular mechanisms comparableto those of calcium, sodium and potassium ions. Zinc ions are simply transported inand out of cells and in and out of cell organelles including vesicular compartments byzinc ion specific transporters, the ZnT and ZIP families (for reviews see Chimientiet al., 2003; Liuzzi and Cousins, 2004; Palmiter and Huang, 2004).
In the mammalian brain, the densest populations of ZEN terminals are found inthe telencephalic structures (Figs. 14a and b). However, zinc-containing terminalsalso invade cerebellum (Figs. 14a and c), hypothalamus, thalamus, mesencephalon,putamen, medulla oblongata, and the spinal cord (Figs. 14d and e) (Perez-Clauselland Danscher, 1985; Perez-Clausell, 1988; Montagnese et al., 1993; Long andFrederickson, 1994; Long et al., 1995; Danscher et al., 2001; Wang et al., 2002a;Valente et al., 2002; Danscher and Stoltenberg, 2005).
Most recently, ZEN vesicles have been found in the peripheral nervous system(PNS) located in axons and somata of a pool of sympathetic neurons by the in vivozinc–selenium AMG technique and by ZnT3 immunohistochemistry (Wang et al.,2002b, 2003).
Until 2001 it was generally believed that ZEN terminals in the CNS wereglutaminergic, but since then it has been proved that in the spinal cord of mice andrats the majority of ZEN terminals are GABAergic (Wang et al., 2001b; Danscheret al., 2001).
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–13996
Until the beginning of the 1980s, most studies on the differentiated and highlylaminated patterns of ZEN terminals in the telencephalic structures were performedwith Timm modifications that were not specific for zinc ions. However, the findingthat the zinc specific chelator diethyldithiocarbamate could completely abolish theTimm staining under certain conditions (Danscher et al., 1973) led to the zinc specificversion of Timm’s original technique (Danscher, 1981a). The following year the invivo selenium AMG technique was worked out (Danscher, 1982) by extrapolation ofthe finding of a colocalization of silver and selenium in the basement membrane ofnephrotic glomeruli (Danscher, 1981c; Aaseth et al., 1981) and of mercury andselenium in the lysosomes of Purkinje cells from a young woman who died in theMinamata accident (Shirabe, 1979).
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139 97
The finding that selenium was as effective as sulphur in creating AMG traceablemetal containing quantum dots had far more importance than one could haveimagined. Not only could the chemical capturing of zinc ions now take place in theZEN terminals of live animals, it also became possible to trace a retrograde axonaltransport of zinc–selenium nanocrystals from the terminals to the lysosomes of ZENneuronal somata (Figs. 15e and f; Danscher, 1982; Howell and Frederickson, 1990;Christensen et al., 1992a; Danscher, 1994; Danscher and Stoltenberg, 2005). Thereason for this new dimension in zinc research, made available by the seleniumtechnique, is most likely the fact that zinc–selenium quantum dots are more resistantto low pH than are zinc–sulphur quantum dots (Stoltenberg and Danscher, 2000).Intracerebral injection of sodium sulphide therefore cannot be used to trace ZENfiber tract somata, owing to the vulnerability of zinc–sulphur nanocrystals to evenweak acids, but can be used most effectively to trace the release of zinc–sulphurnanocrystals in the synaptic cleft over time (Danscher, 1984a–c; Perez-Clausell andDanscher, 1985, 1986).
Extracellular pools of zinc ions, e.g. in unmineralized bone matrix, semen plasma,secrete in the prostate secretory system, in the epididymidal duct and in theLieberkuhn crypts, can be traced with both the sulphur and the selenium basedtechniques.
The functions of the ZnSeAMG traceable zinc ions in the different ZEN cells arenot completely clear. In pancreatic b-cells it has been found that zinc ions inthe secretory granules function as a kind of glue that makes the insulin moleculescollect in hexameres (Dodson and Steiner, 1998; Derewenda et al., 1989). Inthis way the peptide becomes osmotically ‘‘invisible’’. It could very well be thatsuch a function of zinc ions in ZEN vesicles is the original one, i.e. the synapticvesicles are equipped with transmembrane zinc ion transporters causing theinterior zinc ion pressure to increase, which then leads to an ordered packing of
Fig. 14. (a) Micrograph of a 30-mm-thick sagittal cryostat section of rat brain from an animal
treated ip with sodium selenite and allowed to survive 112h before being sacrificed by a
transcardial perfusion with glutaraldehyde. The framed areas are magnified in (b) and (c).
AMG and toluidine blue. Bar ¼ 5mm. (b) ZEN terminals in the telencephalon, all believed to
be glutaminergic, are highly ordered. The different shades from yellow to black are caused by
the sizes and amounts of ZnSeAMG grains in the ZEN terminals. Small terminals with only one
or two ZEN vesicles stain yellow, while huge ZEN boutons with many zinc-enriched synaptic
vesicles stain black primarily because nearby AMG grains confluence, causing an increased
absorption of light. fd, fascia dentata; sub, subiculum. Bar ¼ 300mm. (c) The cochlear nucleus
stains well; note also the yellow AMG staining of the molecular layer of spinocerebellum. ml,
plexus. Bar ¼ 300mm. (d) Coronal section of rat spinal cord from an animal treated ip with
sodium selenide. Note that the ZEN terminals are almost exclusively located in the gray
matter, though with radiation-stained boutons into the white matter. The framed area is
shown magnified in (e). AMG and toluidine blue. Bar ¼ 500 mm. (e) The huge blue stained
motor neurons and their dendrites are covered by ZEN terminals. Note that there exist at least
three different sizes of ZnSeAMG stained boutons. Bar ¼ 30mm. (Figs. 14a–e from Danscher
and Stoltenberg, 2005).
ARTICLE IN PRESS
Fig. 15. (a) Electron micrograph of ZEN terminals in mouse neocortex. The zinc–sulphur
quantum dots have been silver-enhanced by immersion autometallography. Note the many
non ZEN synaptic vesicles. Bar ¼ 1 mm. (From Danscher et al., 2004). (b) Part of a ZEN
terminal showing that the zinc–sulphur quantum dots are located in a pool of synaptic
vesicles. Bar ¼ 0.2mm. (c) Electron micrograph from a contusion-damaged rat neocortex.
Note that the zinc-enriched synaptic vesicles still contain zinc ions even after this severe brain
trauma (Suh et al., unpublished). Bar ¼ 0.5mm. (d) Ultrastructural visualization of AMG
enhanced zinc–sulphur quantum dots in rat exocrine pancreas, using the in vivo Timm
method. Note that only a pool of the secretory vesicles contains zinc ions, i.e. are ZEN
vesicles. Bar ¼ 1mm. (From Danscher, 1996). (e) Light micrograph of Epon section from the
neocortex of a rat treated ip with 8mg sodium selenite per kg body weight 24 h before being
sacrificed. The AMG loaded neuronal somata are the result of retrograde axonal transport of
zinc–selenium quantum dots. Bar ¼ 30 mm. (From Danscher and Stoltenberg, 2005). (f) EM
picture of the anterior commissure from a rat treated with sodium selenide and allowed to
survive for one hour. ZEN axons contain ZnSeAMG grains (arrows) believed to be transported
anterogradely. Bar ¼ 1 mm. (From Danscher and Stoltenberg, 2005.)
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–13998
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139104
reduction of Se(0) to Se(�II) calls for a great amount of electron transfer in arelatively narrow area – transfer processes that damage or kill cells in this area. Thecombination of cauterization and redox stress results in the observed tissue damageand necrosis, and makes selenosulphate unsuitable for intracranial use.
Selenosulphate-based AMG is a stable and reproducible technique that results inuniformly stained sections of high technical quality, and the selenosulphate moleculeis a suitable choice for a strong and reliable staining of ZEN terminals. However, asselenosulphate is not commercially available and demands some effort to produce,sodium selenide/-selenite is the molecule of choice.
6.8.3. Selenocysteine, selenomethionine and other selenium compounds
Since the introduction of the selenium method (Danscher, 1982), differentselenium compounds including selenocysteine and selenomethionine (O’Toole et al.,1995) have been tested to find the ideal selenium donator for demonstration of ZENterminals as well as retrograde axonal transport of zinc ions (Danscher, 1984a–c;Howell and Frederickson, 1990; Christensen et al., 1992a; Danscher, 1994). Themajor disadvantage of most of the compounds previously investigated has been theirrather high tissue toxicity, causing tissue necrosis and the death of severalexperimental animals upon intracranial injection.
In conclusion: For zinc ion tracing we recommend in vivo treatment with sodiumselenite or sodium selenide depending on purpose. The other, commerciallyavailable, selenium compounds tested all result in a less complete creation ofzinc–selenium nanocrystals, but might nevertheless be of interest for research on themetabolism/toxicology of those selenium containing compounds.
7. Control of specificity and how to differentiate between the different
AMG detectable metals
Timm was the first to introduce chemical differentiation of AMG detectable metalsulphides in tissue sections, and he found that treatment of the sections with a weakacid dissolved zinc-sulphur nanocrystals in the sections (Timm, 1961, 1962). Theneed of ensuring that the zinc AMG techniques demonstrated zinc ions only made itimperative to be able to bind the zinc ions in vivo in such a way that they could notbe transformed to zinc–sulphur or zinc–selenium nanocrystals.
As nobody knows the exact amount of zinc ions in the brain, spinal cord or anyother places where zinc ions are present it was vital that the in vivo blocking wasperformed with a low toxic and highly zinc specific chelating agent. Diethyldithio-carbamate (DEDTC) was found to be ideal for this purpose (Danscher et al., 1973)and opened up for an in vivo chemical control that made it possible to designa zinc specific ‘‘Timm sulphide silver method’’ the so-called ‘‘NeoTimm method’’(Danscher, 1981a).
Other authors have dealt with procedures for chemical differentiation of metals intissue sections as well (Voigt, 1959). Previous studies have described procedures
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139106
whereby the nanocrystals are amplified to visible dimensions (Fig. 16) (Goldschmidtet al., 2004).
Goldschmidt et al. have used the technique to map regional differences inthe brain with the Tl-AMG technique. They found that the staining is inaccordance with the regional K+ transfer coefficients and that the local distributionof the tracer is in accordance with an assumpted activity-dependant neuronaluptake of Tl+.
In conclusion: The technique can reveal highly elaborated patterns of neuronalactivity at both light microscopic and ultrastructural levels and is considered suitablefor mapping neuronal activity and studying brain potassium metabolism.
Fig. 16. Pseudocolored image of thallium uptake in layers Ib to Vb of an 8 kHz column in the
primary auditory cortex of a Mongolian gerbil stimulated with alternating pure tones of 1 and
8 kHz. Arrows in layer Vb point from a central cluster of pyramidal neurons to satellite
clusters. Bar ¼ 50 mm. No correction was made for tissue shrinkage. (From Goldschmidt et
al., 2004).
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139 107
9. AMG enhancement of quantum dot bio-probes
Tracing the fate of single molecules at both light microscopic and ultrastructurallevels is a forceful tool in understanding the dynamics of cellular metabolism. As aprelude of the introduction of these new fluorescence probes it was announced in1995 that such nanocrystals are silver enhanceable (Danscher et al., 1995). Theoptimal techniques for tracing the commercially available quantum dots at lightmicroscopic and ultrastructural levels are in progress (Fig. 17; Danscher et al., inpreparation).
Quantum dot probes are nanometer-sized semiconductors with fluorescentqualities and they are therefore suitable for advanced biological imaging of thetagged molecules (Danscher et al., 1995; Hurtley and Helmuth, 2003). Quantum dots(QD) sized 5–10 nm have been suggested to be more photostable than conventionalfluorophores (Chan et al., 2002; Empedocles and Bawendi, 1999; Wu et al., 2003),but possibly an even more important dimension of these new probes is that they canbe AMG silver-enhanced.
Fig. 17. (a) Four different quantum dots written with their chemical symbols in a gelatin film
and thereafter AMG developed for 60min. The four quantum dots are ‘‘CdSe Core Quantum
of a J774 cell exposed to Qtracker 655 cell labeling kit (Quantum Dot Corp.), and AMG
enhanced for 60min. The Qtracker 655 cell labeling kit contains a CdSe core encapsulated in a
shell of ZnS. The quantum dot is conjugated to a membrane translocating molecules (MTM);
details from the manufacturer is not available. Bar ¼ 2mm.
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139 109
(b)
1. Citrate buffer (pH 3.7): Dissolve 25.5 g citric acid 1 �H2O and 23.5 g trisodiumcitrate 2 �H2O in deionized water to make 100ml.
2. Lactate buffer (pH 3.8): Dissolve 31.5ml of 50% sodium lactate and 6ml of90% lactic acid in deionized water to make 100ml.
(c)
Hydroquinone: Dissolve 0.85 g hydroquinone in 15ml deionized water. Preparejust before use.
(d)
Silver ion supply: Dissolve 0.12 g silver lactate in 15ml deionized water. Thesolution should be protected from light by wrapping tin folio around the vial.
The silver lactate, hydroquinone and citrate buffer solutions have to be heated to40 1C for the ingredients to dissolve completely.
Mix solution a (60ml), solution b1 or b2 (10ml), and solution c (15ml) carefully ina 100ml beaker. Add solution d (15ml) immediately before use and mix thoroughly.
The glass slides placed in 10% Farmer cleaned jars are poured with the AMGdeveloper, the jars are placed in a water bath on a shaker, set at 26 1C, and a lighttight hood is placed over the set-up.
10.1.3. Cellulose silver lactate
a.
60ml 2% cellulose (carboxymethylcellulose, Sigma C-4888) b. 10ml sodium citrate buffer or lactate buffer c. Dissolve 0.85 g hydroquinone in 15ml deionized water d. Dissolve 0.12 g silver lactate in 15ml deionized water e. 21
2ml thiosulphate (0.625 sodium thiosulphate in 100ml distilled water)
The 2% cellulose is dissolved in distilled water 1–2 days before use and kept freshfor at least two months.
If wanted, the reducing molecule hydroquinone can be replaced by the electrondonator N-Propyl gallat and silver lactate with silver acetate without changing theoverall quality of the Cellulose AMG Developer.
It is important to use white silver lactate powder when preparing the solution andit should be used immediately. If the container is wrapped in a metal foil and keptcool it can be stored for an hour. If the powder is brownish the development will takemore time, i.e., there are less available silver ions for silver enhancement.
It is also important to ensure that the hydroquinone solution is used immediatelyafter being prepared and that the temperature of the solution must not at any timeexceed 45 1C. The citrate buffer can keep fresh for more than a year.
The length of development that is needed to obtain a certain degree of silverenhancement can be controlled by (1) regulating the temperature, (2) regulating thelevel of reductor molecules, (3) modifying the pH, and (4) changing the amount ofavailable silver ions.
Stierhof et al. have meticulously analysed the quality of some of the most usedAMG developers and their articles contain important discussions of the principles ofsilver enhancement (Stierhof et al., 1991).
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139110
10.1.4. Silver acetate developer (Hacker et al., 1990)
1.
Rinse carefully about eight times in deionized or glass distilled water. 2. Solution A: Dissolve 100mg silver acetate in 50ml of deionized and glass distilled
water. Fresh solutions must be made for every run. Silver acetate crystals can bedissolved easily by continuous stirring for about 15min, provided that finecrystals are used. Larger crystals may be dissolved in an ultrasonic bath. It isadvisable to filter solution A shortly before use.
3.
Citrate buffer, pH 3.8: Dissolve 25.5 g citric acid 1 �H2O and 23.5 g trisodiumcitrate 2 �H2O in distilled water to make 100ml. Adjust to pH 3.8 with citric acid.This buffer can be kept at 4 1C for at least 2–3 weeks. Before use, check the pH.
4.
Solution B: Dissolve 250mg Hq in 50ml citrate buffer. 5. Just before use, mix solutions A and B. 6. Silver amplification: Cover the slides with the mixture of solutions A and B.
Staining intensity may be checked with the light microscope during theamplification process, which is stopped by immersion in the photographic fixer.
7.
The photographic fixer (e.g., Ilfospeed or Agefix) diluted 1:10 may be used to stopthe amplification process immediately. This solution can be re-used for manystainings. Leave the slides in this solution for about 1min. Alternatively, use a 5%aqueous solution of sodium thiosulphate.
8.
Rinse the slides carefully in tap water for at least 3min. After silver amplification,the sections can be counterstained with hematoxylin or nuclear fast red,dehydrated, and mounted in DEPEX.
The use of silver acetate AMG allows development to take place undernormal laboratory daylight conditions, whereas the silver lactate developmentshould preferably take place under a black cover at the laboratory bench.The acid silver acetate developer allows the AMG process to be controlledvisually with a normal bright-field microscope and interrupted at the optimumlevel (as does the silver lactate developer if the developing time is below 30minutes).
10.1.5. After AMG development
Developing time: After the appropriate developing time (20–60min, dependingon the thickness of the sections) the AMG developer is replaced with a 5%sodium thiosulphate solution, and after 10min the jars are placed in runningtap water. To control the efficiency of the developer a control slide with sectionsthat demonstrate an easy-to-judge AMG staining pattern is placed in each jar.This is an easy and thoroughly tested way of knowing if anything has gonewrong.
Counterstaining or e.g. immunohistochemical staining can now be performed.After dehydration, the sections can be mounted with DEPEX or any other non-acidresin.
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139 115
It is imperative that the zinc ion pool in the synaptic vesicles of ZEN neuronalterminals is chemically bound as stable crystals before it can escape from the vesicles.In tissue sections, the only way of making sure that the zinc ions are trapped and staylocalized is to keep the tissue frozen until the chemical binding has taken place. It istherefore of the utmost importance that the biopsy material is frozen as soon aspossible in liquid nitrogen or by CO2 gas and kept frozen until the zinc ions arecaptured by sulphide ions in the gas chamber. However, when tissue has been frozen, itcan be stored for an extended period of time with only minor leakage of zinc ions and,as a result, almost no decrease in the quality of the AMG staining. AMG analysis ofhuman brains 6 h post mortem has given most unsatisfactory results because of thediffusion of zinc ions from the zinc ion containing synaptic vesicles of ZEN boutons, aprocess that most likely starts as soon as the terminals are deprived of oxygen.Therefore, only biopsies from surgical or diagnostic intervention in the human braincan be expected to give an optimal AMG demonstration of the ZEN terminals.However, the possibility that human brain autopsy specimens taken shortly afterdeath still contain appreciable amounts of sulphide-available zinc ions in their ZENterminals cannot be excluded human brain autopsy specimens taken shortly afterdeath still contain appreciable amounts of sulphide-available zinc ions in their ZENterminals (Danscher and Zimmer, 1978). In rats, a usable AMG staining of the ZENterminals can be obtained from animals that have died 2h before the brain is removed.
10.6.2. Handling of tissue sections
Biopsies from human patients are frozen in liquid nitrogen immediately afterremoval in connection with surgical intervention. Likewise, brains and other organsremoved from deeply anesthetized or decapitated Wistar rats are frozen on removal.These biopsies and tissue blocks must be kept at �80 1C.
The frozen tissue blocks are glued to a cryostat stage by Tissue-Tek OCT 4583Compound (Miles; Elkhart, IN), placed in a cryostat, and allowed to increase intemperature to �17 1C. Then 30-mm-thick sections are cut and mounted on 10%Farmer cleaned glass slides. The sections are thawed for a few seconds to adhere tothe slide and then stored at �80 1C until moved to the sulphidation chamber. Thesections are placed in the chamber for 30min before being flushed with H2S gas for2min, followed by 5min of intermission and another 2min of H2S flushing. Gas flowis adjusted to a level at which vivid bubbling is seen in the washing bottles.
The exposure time in the now H2S-filled chamber varies from 1 to 24 h. In mostcases an exposure period of 6–8 h has been found to be optimal, but in order not tomiss any traces of zinc ions in the sections it is advisable to expose initially for 24 h.
After H2S exposure, the chamber is flushed with nitrogen gas for 10min. Then thechamber is opened and the cradles removed and placed in 70% alcohol for 30min.Later, the sections are rehydrated and finally dipped in a 0.5% gelatin solution.
Cryostat sections to be analysed in the electron microscope are cut, 50–70 mmthick, and placed in a small (volume 8ml) teflon bowl kept at �17 1C. The bowl istransferred to the gas chamber. After H2S exposure it is filled with cold 3%glutaraldehyde in a 0.1M phosphate solution. The sections are allowed to thaw for30min in this solution at room temperature, rinsed in distilled water three times,
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139 117
(2)
Paraffin sections: The slices are embedded in paraffin while checking that thetemperature during the embedding does not exceed 45 1C, cut into 5-mm-thicksections and placed on glass slides. These are AMG developed, counterstainedwith toluidine blue and mounted in DEPEX.
(3)
Epon sections: The small blocks cut out from the slices are embedded in Eponwhile keeping the polymerization temperature below 45 1C. From the Eponblocks 3-mm sections are cut, placed on glass slides and AMG developed. AfterLM analysis, sections to be analysed in the electron microscope are reembeddedon top of an Epon block and from these preparations ultrathin sections are cut,placed on a grid and counterstained with uranyl citrate and lead acetate.
10.7.5. AMG development of NTS slices
The slices are placed in 10% Farmer cleaned jars and poured with the AMGdeveloper. The jars are placed in a water bath at 26 1C and covered by a light tighthood. After 60min the development is stopped by replacing the developer with 5%sodium thiosulphate. After 10min the slices are rinsed carefully for 5min in severalwashes of distilled water.
10.7.6. Post AMG treatment of NTS slices
Paraffin sections: The slices are embedded in paraffin without temperature controland 5-mm-thick sections are cut, placed on glass slides, counterstained with toluidineblue and mounted in DEPEX.
Epon sections: The areas to be analysed in the electron microscope are dissectedfrom the AMG developed slices and these small blocks of tissue are placed in a vialwhere they are fixed in 0.1% osmium tetroxide for 30min. After proper rinsing indistilled water, the sections are embedded in Epon without temperature control.From these Epon blocks, 3-mm-thick sections are cut and analysed in the lightmicroscope. The sections to be analysed are then reembedded on top of a blankEpon block, and after proper trimming ultrathin sections are cut and stainedsecundum artem.
10.7.7. Human tissue
Human tissues obtained from autopsies or biopsies are fixed in 4% bufferedformalin for 2 h before being immersed in the NTS for 3 days. Sections for light andelectron microscopical analyses are performed as described above. If the tissueblocks are less than 2mm in diameter they can be placed in the NTS without priorcutting.
10.7.8. NTS based iZnSAMG specificity for zinc
The above optimal condition for iZnSAMG has been tested for zinc specificity intwo different ways: (1) Animals have been treated with 1000mg/kg animal weight ofthe low toxic zinc chelator diethyldithiocarbamate (DEDTC) one hour beforesacrifice, and the brains and other zinc ion containing tissues have been treated in theabove optimum standard way. In sections from these sources we found no staining at
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139 123
To obtain the final GGS, 8 g gelatin powder (Merck 4078) is dissolved in 60mldistilled water. To this solution is added 40ml of the gold solution and the mixture isheated to 40 1C at constant stirring.
11. Epilogue
At the moment we know that tissue sections from animals that have been exposedto lead, copper, cadmium, iron and aluminium in vivo do not contain AMG grainsafter development although these metals can by traced in the tissues by both atomicabsorption spectrophotometry (AAS) and the multielement analysis approachPIXE. It is, on the other hand, possible to trace lead–sulphur, copper–sulphur,iron–sulphur and aluminum–sulphur nanocrystals or the corresponding seleniumquantum dots by AMG if they are present in a gelatin layer on an object glass. Thistells us that these metals are not metabolized into their respective metal–sulphur/–selenium quantum dots in the organism (Danscher, unpublished).
If rats and mice that have been exposed to one of the above mentioned metals areconcomitantly treated with sodium selenide or after a period of time perfused withsodium sulphide, no ‘‘extra’’ staining, i.e. staining not caused by zinc–sulphurrespectively zinc–selenium nanocrystals, has been traced. This suggests that Pb-, Cu-,Cd-, Fe- and Al-S/Se nanocrystals are not created in tissue sections (Danscher, 1988;Danscher, unpublished). However, several authors have reported that they haveobserved AMG grains that they judge are caused by nanocrystals containing copper,cadmium, iron, aluminium, or lead after having exposed the tissue or cultures to highconcentrations of sulphide ions at high pH values (pH 10 or more) (e.g. Brunk andBrun, 1972; Zdolsek et al., 1993; Soto et al., 1996; Yuan, 1999; Domouhtsidou andDimitriadis, 2000). We suggest that these results may reflect that an extreme pH or aspecial fixative may cause some otherwise firmly bound metal ions to becomeavailable for the creation of catalytic nanocrystals, or they may be the result ofthe excess amount of sulphide ions used. Such a surplus of sulphide ions might bindto different radicals in the tissue and create silver–sulphur nanocrystals when placedin the AMG developer – such nanocrystals will in turn be silver-enhanced and lookas genuine AMG grains (Danscher, 1981a). The only way to close this still opendispute is to collect AMG grains from sections or cultures by removing the organicmaterial enzymatically and analyse their metallic content with a multielementanalysis, e.g. PIXE.
As stressed several times in this article, gold is bound in cells that have beenexposed to a gold salt, e.g. aurothiomalate, in a way that does not allow it to betraced directly by AMG. A transformation of chemically bound gold ions to metallicgold atoms is needed before they can be traced with AMG. This suggests that goldions are not metabolized into gold–sulphur/–selenium quantum dots, but are treatedmetabolically different from, e.g., silver, bismuth, and mercury that all end up insulphur-containing nanocrystals in the living organism.
The possibility that heavy metals that have not yet been tested with the AMGapproach will be metabolized through the same or equivalent routes as silver,
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139124
mercury and bismuth and end up as catalytic metal–sulphur/–selenium nanocrystalscannot at all be excluded (Danscher et al., 1995, 2002).
That copper sulphide clusters can be AMG silver-enhanced is well known as it isused in the cuprolinic blue technique for visualizing copper dyes in glycosaminogly-cans (Lormee et al., 1989). As such nanocrystals, however, do not seem to be createdin the living organism, neither after in vivo treatment with sodium selenide nor afterperfusion with sodium sulphide, we found the inclusion of a protocol for dissolvingcopper–sulphur/–selenium quantum dots irrelevant. It is, however, very easy toperform.
The elegant use of thallium as a tool for high-resolution mapping of neuronalactivity, by crystallizing thallium taken up by activated neurons in Tl-S quantumdots, is noteworthy and promises well for future applications of the AMG technique.There is little doubt that the AMG technology will be further sophisticated in theyears to come and that new AMG nanocrystals will be introduced as valued tools inbiology and other scientific fields.
The list of reprint permission acknowledgements
Fig. 2
Permission granted by Elsevier Ltd. to use:Fig. 2a from the following:
Title of journal:
Progress in Histochemistry and Cytochemistry Author: G. Danscher Volume: 23 Issue: (1–4) Year: 1991 Pages: 273–285 Article title: Histo- and Cytochemistry as a Tool in Environmental
Toxicology. Histochemical tracing of zinc, mercury, silver andgold
Fig. 3a
Permission granted by Elsevier Ltd. to use:Part of Fig. 1 (close up of the cell marked Ga)from the following:
Title of journal:
Experimental and Molecular Pathology Author(s): Schiønning, J.D., E.H. Poulsen et al. Volume: 56 Issue: 3 Year: 1992 Pages: 239–247 Article title: Autometallographic detection of gold in Dorsal Root Ganglia of
Rats Treated with Sodium Aurothiomalate
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139 125
Fig. 3b and c
Permission granted by Springer Science and Business Media to use:Fig. 1B and 2A from the following:
Title of journal:
Histochemistry and Cell Biology Author: Gorm Danscher Volume: 117 Issue: (5) Pages: 447–452 Year: 2002 Article title: In vivo liberation of gold ions from gold implants.
Autometallographic tracing of gold in cells adjacent to metallicgold.
Fig. 3d
Permission granted by The Journal of Histochemistry and Cytochemistry to use:Fig. 4 from the following:
Title of journal:
The Journal of Histochemistry and Cytochemistry Author(s): G. Danscher, J. O. R. Norgaard Volume: 31 Issue: (12) Pages: 1394-1398 Year: 1983 Article title: Light Microscopic Visualization of Colloidal Gold on Resin-
embedded Tissue
Fig. 4a–d
Permission granted by the Journal of Histochemistry and Cytochemistry to use:Fig. 1a–d from the following:
Title of journal:
Journal of Histochemistry and Cytochemistry Author(s): G. Danscher, J.O.R. Norgaard Volume: 33 Issue: 7 Pages: 706–710 Year: 1985 Article title: Ultrastructural Autometallography: A Method for Silver
Amplification of Catalytic Metals
Fig. 5a and b
Permission granted by Elsevier Ltd. to use:Fig. 2b and c from the following:
Title of journal:
Journal of Neuroscience Methods Author(s): Danscher,G., A. Andreasen
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139126
Volume:
77 Issue: (2) Year: 1997 Pages: 175–181 Article title: Demonstration of vessels in CNS and other organs by AMG
silver enhancement of colloidal gold particles dispersed ingelatine
Fig. 7a–b
Permission granted by Springer Science and Business Media to use:Fig. 2 and 8 from the following:
Title of journal:
Histochemistry Author: Gorm Danscher Volume: 71 Issue: (2) Pages: 177–186 Year: 1981 Article title: Light and Electron Microscopic Localization of Silver in
Biological Tissue
Fig. 10
Permission granted by Taylor & Francis Group to use:Fig. 8 from the following:
Title of journal:
Stain Technology – now Biotechnic and Histochemistry Author: Gorm Danscher Volume: 58 Issue: (6) Pages: 365–372 Year: 1983 Article title: A Silver Method for Counterstaining Plastic Embedded Tissue
Fig. 11a
Permission granted by Elsevier Ltd. to use:Fig. 2 from the following:
Title of journal:
Progress in Histochemistry and Cytochemistry Author(s): P. Horsted-Bindslev, J.E. Bolewska et al. Volume: 23 Issue: (1–4) Year: 1991 Pages: 321–326 Article title: Dentinal transport of mercury from dental silver amalgam
restorations
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139 127
Fig. 11c and 11d
Permission granted by the Journal of Histochemistry and Cytochemistry to use:Figs. 14 and 15 from the following:
Title of journal:
The Journal of Histochemistry and Cytochemistry Author(s): G. Danscher, B. Møller-Madsen Volume: 33 Issue: 3 Pages: 219–228 Year: 1985 Article title: Silver Amplification of Mercury Sulfide and Selenide:
A Histochemical Method for Light and Electron MicroscopicLocalization of Mercury in Tissue
Fig. 12
Permission granted by Elsevier Ltd. to use:Fig. 2b and 2c from the following:
Title of journal:
Progress in Histochemistry and Cytochemistry Author: G. Danscher Volume: 23 Issue: (1-4) Year: 1991 Pages: 273–285 Article title: Histo- and Cytochemistry as a Tool in Environmental
Toxicology. Histochemical tracing of zinc, mercury, silver andgold
Fig. 13c
Permission granted by Taylor & Francis Group to use:Fig. 3 from the following:
Title of journal:
Scandinavian Journal of Gastroenterology Author(s): M. Stoltenberg, M. Martiny, K. Sørensen, J. Rungby and
K.A. Krogfeldt
Volume: 36 Issue: (2) Pages: 144–148 Year: 2001 Article title: Histochemical Tracing of Bismuth in Helicobacter pylori after In
Vitro Exposure to Bismuth Citrate
Fig. 14a–e and 15e–f
Permission granted by Journal of Histochemistry and Cytochemistry to use:Figs. 1a, 1b, 1d, 2a, 2b, 4a and 4d from the following:
Title of journal:
Journal of Histochemistry and Cytochemistry
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139128
Author(s):
G. Danscher, M. Stoltenberg Volume: 53 Issue: (2) Pages: 141–153 Year: 2005 Article title: Zinc-specific Autometallographic In Vivo Selenium Methods:
Tracing of Zinc-enriched (ZEN) Terminals, ZEN Pathways, andPools of Zinc Ions in a Multitude of Other ZEN Cells.
Fig. 15a
Permission granted by the Journal of Histochemistry and Cytochemistry:Fig. 2 from the following:
Title of journal:
Journal of Histochemistry and Cytochemistry Author(s): G. Danscher, M. Stoltenberg, M. Bruhn et al. Volume: 52 Issue: (12) Pages: 1619–1625 Year: 2004 Article title: Immersion Autometallography: Histochemical In Situ Capturing
of Zinc Ions in Catalytic Zinc–Sulphur Nanocrystals
Fig. 15d
Permission granted by Springer Science and Business Media to use:Fig. 5b (close up photo)from the following:
Title of journal:
Histochemical Journal Author: Gorm Danscher Volume: 28 Issue: (5) Pages: 361–373 Year: 1996 Article title: The autometallographic zinc–sulphide method. A new approach
involving in vivo creation of nanometer-sized zinc sulphidecrystal lattices in zinc-enriched synaptic and secretory vesicles.
Fig. 16
Permission granted by Elsevier Ltd. to use:Fig. 6 from the following:
Title of journal:
NeuroImage Author(s): Jurgen Goldschmidt, W. Zuschratter, H. Scheich Volume: 23 Issue: (2) Year: 2004
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139 129
Pages:
638–647 Article title: High-resolution mapping of neuronal activity by thallium
Acknowledgements
The authors gratefully acknowledge the skilful laboratory technical assistanceof Ms. D. Jensen, Ms L. Munkøe and Ms M. Sand. Special thanks toMs K. Wiedemann for her high-quality, professional work on the manuscript andto Mr. A. Meier for his excellent photographic input. Also we thank Dr. Søren Juhlfor synthesizing the selenosulfate and Dr. Agnete Larsen for her help with pilotscreenings of the ‘‘quantum-dot-tracer-autometallography’’ concept and use of tat-nanogold as a marker of isolated cells to be tracked in a recipient organism. Thestudy was supported by The Danish Medical Research Council, The AarhusUniversity Research Foundation, The Health Science Faculty, The Aase & EjnarDanielsen Foundation, The Danish Medical Association Research Fund, TheLundbeck Foundation, The Leo Foundation, The Beckett Foundation, TheGangsted Foundation, The Augustinus Foundation and The Novo NordicFoundation.
References
Aaseth J, Olsen A, Halse J, Hovig T. Argyria-tissue deposition of silver as selenide. Scand J Clin LabInvest 1981;41(3):247–51.
Arenholt-Bindslev D, Danscher G. Effect of organic and inorganic selenium on mercury accumulation incultures of normal human epithelial cells. ATLA (Alternative to Laboratory Animals) 1989;16:253–6.
Arvidson B. Inorganic mercury is transported from muscular nerve terminals to spinal and brainstemmotor neurons. Muscle Nerve 1992;15:1089–94.
Baatrup E, Danscher G. Cytochemical demonstration of mercury deposits in trout liver and kidneyfollowing methyl mercury intoxication. Differentiation of two mercury pools by selenium. EcotoxEnviron Safety 1987;14:129–41.
Bendayan M. Ultrastructural localization of nuclei acids by the use of enzyme–gold complexes.J Histochem Cytochem 1981;29(4):531–41.
Birinyi A, Parker D, Antal M, Shupliakov O. Zinc co-localizes with GABA and glycine in synapses in thelamprey spinal cord. J Comp Neurol 2001;433(2):208–21.
Bolewska J, Holmstrup P, Møller-Madsen B, Kenrad B, Danscher G. Amalgam associated mercuryaccumulations in normal oral mucosa, oral mucosal lesions of lichen planus and contact lesionsassociated with amalgam. J Oral Pathol Med 1990;19:39–42.
Britten JS, Blank M. Thallium activation of the (Na+–K+)-activated ATPase of rabbit kidney. BiochimBiophys Acta 1968;159(2):160–6.
Brown CE, Dyck RH. An improved method for visualizing the cell bodies of zincergic neurons. J NeurosciMethods 2003;129(1):41–7.
Brunk U, Brun A. Histochemical evidence for lysosomal uptake of lead in tissue cultured fibroblasts.Histochemie 1972;29:140–6.
Cassell MD, Brown MW. The distribution of Timm’s stain in the nonsulphide-perfused humanhippocampal formation. J Comp Neurol 1984;222:461–71.
Chan WC, Maxwell DJ, Gao X, Bailey RE, Han M, Nie SW. Luminescent quantum dots for multiplexedbiological detection and imaging. Curr Opin Biotechnol 2002;13(1):40–6.
Cheung AL, Graf AH, Hauser-Kronberger C, Dietze O, Tubbs RR, Hacker GW. Detection of humanpapillomavirus in cervical carcinoma: comparison of peroxidase, Nanogold, and catalyzed reporterdeposition (CARD)-Nanogold in situ hybridization. Mol Pathol 1999;12(7):689–96.
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139130
Chimienti F, Aouffen M, Favier A, Seve M. Zinc homeostasis-regulating proteins: new drug targets fortriggering cell fate. Curr Drug Targets 2003;4(4):323–38.
Christensen M- K, Frederickson CJ, Danscher G. Retrograde tracing of zinc-containing neurons byselenide ions: A survey of seven selenium compounds. J Histochem Cytochem 1992a;40:575–9.
Christensen MM, Danscher G, Ellermann-Eriksen S, Schiønning JD, Rungby J. Autometallographicsilver-enhancement of colloidal gold particles used to label phagocytic cells. Histochemistry1992b;97:207–11.
Cohen-Kfir E, Lee W, Eskandari S, Nelson N. Zinc inhibition of gamma-aminobutyric acid transporter 4(GAT4) reveals a link between excitatory and inhibitory neurotransmission. Proc Natl Acad Sci USA2005;102(17):6154–9.
Dahan M, Level S, Luccardini C, Rostaing P, Riveau B, Triller A. Diffusion dynamics of glycine receptorsrevealed by single-quantum dot tracking. Science 2003;302(5644):442–5.
Daneels G, Moeremans M, De Raeymaeker M, De Mey J. Sequential immunostaining (gold/silver) andcomplete protein staining (AuroDye) on Western blots. J Immunol Methods 1986;89(1):89–91.
Danscher G. Ultrastructural localization of metals in the CNS by physical development: Gold, mercury,and water-insoluble metal sulfides. J Ultrastr Res 1980;73(1):93 (abstract).
Danscher G. Histochemical demonstration of heavy metals. A revised version of the sulphide silvermethod suitable for both light and electron microscopy. Histochemistry 1981a;71:1–16.
Danscher G. Localization of gold in biological tissue. a photochemical method for light andelectronmicroscopy. Histochemistry 1981b;71:81–8.
Danscher G. Light and electron microscopic localization of silver in biological tissue. Histochemistry1981c;71:177–86.
Danscher G. Exogenous selenium in the brain. A histochemical technique for light and electronmicroscopical localization of catalytic selenium bonds. Histochemistry 1982;76:281–93.
Danscher G. A silver method for counterstaining plastic embedded tissue. Stain Technol 1983;58:365–72.Danscher G. Similarities and differences in the localization of metals in rat brains after treatment with
sodium sulphide and sodium selenide. In: Frederickson CJ, Howell GA, Kasarkis E, editors. Theneurobiology of zinc, part A. New York: Alan R. Liss; 1984a. p. 229–42.
Danscher G. Do the timm sulphide silver method and the selenium method demonstrate zinc in the brain.In: Frederickson CJ, Howell GA, Kasarkis E, editors. The neurobiology of zinc, part A. New York:Alan R. Liss; 1984b. p. 273–87.
Danscher G. Dynamic changes in the stainability of rat hippocampal mossy fiber boutons after localinjection of sodium sulphide, sodium selenite, and sodium diethyldithiocarbamate. In: FredericksonCJ, Howell GA, Kasarkis E, editors. The neurobiology of zinc, part B. New York: Alan R. Liss; 1984c.p. 177–91.
Danscher G. Autometallography. A new technique for light and electron microscopic visualization ofmetals in biological tissue (gold, silver, metal sulphides and metal selenides). Histochemistry1984d;81:331–5.
Danscher G. Can aluminium be visualized in CNS by silver amplification? Acta Neuropathol (Berl)1988;76(1):107.
Danscher G. Histochemical tracing of zinc, mercury, silver and gold. Progr Histochem Cytochem1991;23:273–85.
Danscher G. Autometallographic (AMG) nerve tracing: demonstration of retrograde axonal transport ofzinc selenide in zinc-enriched (ZEN) neurons. In: Gu J, Hacker GW, editors. Modern methods inanalytical morphology. New York: Plenum Press; 1994. p. 327–39.
Danscher G. The autometallographic zinc-sulphide method. A new approach involving in vivo creation ofnanometer-sized zinc sulphide crystal lattices in zinc-enriched synaptic and secretory vesicles.Histochem J 1996;28:361–73.
Danscher G. In vivo liberation of gold ions from gold implants. Autometallographic tracing of gold incells adjacent to metallic gold. Histochem Cell Biol 2002;117:447–52.
Danscher G, Andreasen A. Demonstration of vessels in CNS and other organs by AMG silverenhancement of colloidal gold particles dispersed in gelatine. J Neurosci Meth 1997;77:175–81.
Danscher G, Møller-Madsen B. Silver amplification of mercury sulfide and selenide. A histochemicalmethod for light and electron microscopic localization of mercury in tissue. J Histochem Cytochem1985;33:219–28.
Danscher G, Montagnese C. Autometallographic localization of synaptic vesicular zinc and lysosomalgold, silver and mercury. J Histotechnol 1994;17:15–22.
Danscher G, Nørgaard JOR. Light microscopic visualization of colloidal gold on resin-embedded tissue.J Histochem Cytochem 1983;31/12:1394–8.
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139 131
Danscher G, Rungby. Differentiation of histochemically visualized mercury and silver. Histochem J1986;18:109–14.
Danscher G, Nørgaard JOR. Ultrastructural autometallography: a method for silver amplification ofcatalytic metals. J Histochem Cytochem 1985;33(7):706–10.
Danscher G, Stoltenberg M. 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.J Histochem Cytochem 2005;53(2):141–53.
Danscher G, Zimmer J. An improved Timm sulphide silver method for light and electron microscopiclocalization of heavy metals in biological tissues. Histochemistry 1978;55:27–40.
Danscher G, Haug F- MS, Fredens K. Effect of diethyldithiocarbamate (DEDTC) on sulphide silverstained boutons. Reversible blocking of Timm’s sulphide silver stain for ‘‘heavy’’ metals in DEDTCtreated rats (light microscopy). Exp Brain Res 1973;16:521–32.
Danscher G, Hansen HJ, Møller-Madsen B. Energy dispersive X-ray analysis of tissue gold after silveramplification by physical development. Histochemistry 1984;81:283–5.
Danscher G, Howell G, Perez-Clausell J, Hertel N. The dithizone, Timm’s sulphide silver and the seleniummethods demonstrate a chelatable pool of zinc in CNS. Histochemistry 1985a;83:419–22.
Danscher G, Thorlacius-Ussing O, Rungby J, Møller-Madsen B. Selenium in the Paneth cells. Sci TotalEnviron 1985b;42:189–92.
Danscher G, Hørsted-Bindslev P, Rungby J. Traces of mercury in organs from primates with amalgamfillings. Exp Mol Pathol 1990;52:291–9.
Danscher G, Hacker G, Nørgaard JOR, Grimelius L. Autometallographic silver amplification of colloidalgold. J Histotechnol 1993;16:201–7.
Danscher G, Stoltenberg M, Juhl S. How to detect gold, silver and mercury in human brain and othertissues by autometallographic silver amplification. Neuropathol Appl Neurobiol 1994;20:454–67.
Danscher G, Hacker GW, Hauser-Kronberger C, Grimelius L. Trends in autometallographic silveramplification of colloidal gold particles. In: Hayat MA, editor. Immunogold-silver staining. Principles,methods, and applications. Boca Raton: CRC Press; 1995. p. 11–8.
Danscher G, Jensen KB, Kraft J, Stoltenberg M. Autometallographic silver enhancement ofsubmicroscopic metal containing catalytic crystallites – a histochemical tool for detection of gold,silver, bismuth, mercury, and zinc. Cell Vision 1997a;4:375–86.
Danscher G, Juhl S, Stoltenberg M, Krunderup B, Schrøder HD, Andreasen A. Autometallographic silverenhancement of zinc sulfide crystals created in cryostat sections from human brain biopsies. A newtechnique that makes it feasible to demonstrate zinc ions in tissue sections from biopsies and earlyautopsy material. J Histochem Cytochem 1997b;45:1503–10.
Danscher G, Mosekilde L, Rungby J. Histochemical detection of zinc in mineralizing rat bone:autometallographic tracing of zinc ions in the mineralization front, osteocytes, and osteoblasts.J Histotechnol 1999;22:85–90.
Danscher G, Stoltenberg M, Kemp K, Pamphlett R. Bismuth autometallography. Protocol – specificity –differentiation. J Histochem Cytochem 2000;48:1503–10.
Danscher G, Jo SM, Varea E, Wang Z, Cole TB, Schrøder HD. Inhibitory zinc-enriched terminals inmouse spinal cord. Neuroscience 2001;105(4):941–7.
Danscher G, Hacker GW, Stoltenberg M. Autometallographic tracing of gold, silver, bismuth, mercury,and zinc. In: Hacker GW, Gu J, editors. Gold and Silver Staining. Techniques in MolecularMorphology. Boca Raton: CRC Press; 2002.
Danscher G, Stoltenberg M, Bruhn M, Søndergaard C, Jensen D. Immersion autometallography –iZnSAMG: histochemical in situ capturing of zinc ions in catalytic zinc–sulphur nanocrystals.J Histochem Cytochem 2004;52(12):1619–25.
de la Fuente JM, Berry CC. Tat peptide as an efficient molecule to translocate gold nanoparticles into thecell nucleus. Bioconjug Chem 2005;16(5):1176–80.
Derewenda U, Derewenda Z, Dodson GG, Hubbard RE, Korber F. Molecular structure of insulin: theinsulin monomer and its assembly. Br Med Bull 1989;45(1):4–18.
Dodson G, Steiner D. The role of assembly in insulin’s biosynthesis. Curr Opin Struct Biol1998;8(2):189–94.
Domouhtsidou GP, Dimitriadis VK. Ultrastructural localization of heavy metals (Hg, Ag, Pb, and Cu) ingills and digestive gland of mussels, Mytilus galloprovincialis (L.). Arch Environ Contam Toxicol2000;38:472–8.
Dore JL. The demonstration and demonstration and distribution of gold in tissue sections. Thesis(London) 1974.
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139132
Dore JL, Vernon-Roberts BA. A method for the selective demonstration of gold in tissue sections. MedLab Sci 1976;33:209–13.
Douglas KT, Bunni MA, Baindur SR. Thallium in biochemistry. Int J Biochem 1990;22:429–38.Duong TQ, Kim D- S, Ugurbil K, Kim S- G. Localized cerebral blood flow response at submillimeter
columnar resolution. Proc Natl Acad Sci USA 2001;98:10904–9.Empedocles S, Bawendi M. Spectroscopy of single CdSe nanocrystallites. Acc Chem Res 1999;
32(5):389–96.Faulk WP, Taylor GM. An immunocolloid method for the electron microscope. Immunochemistry
1971;8(11):1081–3.Fowler BA, Brown HW, Lucier GW, Bread ME. Mercury uptake by renal lysosomes of rats ingesting
methyl mercury hydroxide. Arch Pathol 1974;98:297–301.Frederickson CJ, Danscher G. Zinc-containing neurons in hippocampus and related CNS structures. Prog
Brain Res 1990;83:71–84.Frederickson CJ, Perez-Clausell J, Danscher G. Zinc-containing 7S-NGF complex. Evidence from zinc
histochemistry for localization in salivary secretory granules. J Histochem Cytochem 1987;35(5):579–83.
Frederickson CJ, Suh SW, Silva D, Frederickson CJ, Thompson RB. Importance of zinc in the centralnervous system: the zinc-containing neuron. J Nutr 2000;130(5S Suppl):1471S–83S.
Fujimori O. A double protein A–gold-silver staining method for tissue antigens in light microscopy.Histochem J 1992;24(2):61–6.
Fujimori O, Nakamura M. Protein A gold-silver staining method for light microscopic immunohis-tochemistry. Arch Histol Japan 1985;48(4):449–52.
Fujimori O, Sobue HM, Yamada K. Lectin binding sites of glycoproteins as revealed by lectin–gold–silverand lectin-peroxidase-diaminobenzidine methods. Histochem J 1988;20(11):603–9.
Gallyas F. A principle for silver staining of tissue elements by physical development. Acta Morphol AcadSci Hung 1971;19(1):57–71.
Gallyas F. Factors affecting the formation of metallic silver and the binding of silver ions by tissuecomponents. Histochemistry 1979a;64(1):97–109.
Gallyas F. Light insensitive physical developers. Stain Technol 1979b;54(4):173–6.Gallyas F. An argyrophil III method for the demonstration of micro- and oligodendroglia. Acta Morphol
Acad Sci Hung 1981;29(2–3):177–83.Gallyas F, Guldner FH, Zoltay G, Wolff JR. Golgi-like demonstration of ‘‘dark’’ neurons with an
argyrophil III method for experimental neuropathology. Acta Neuropathol (Berl) 1990;79(6):620–8.Gehring PJ, Hammond PB. The interrelationship between thallium and potassium in animals.
J Pharmacol Exp Ther 1967;155:187–201.Gilg E. A photochemical method for microdetection of gold in tissue sections. Acta Psychiatr Scand
1952;27:43–56.Goldschmidt J, Zuschratter W, Scheich H. High-resolution mapping of neuronal activity by thallium
autometallography. Neuroimage 2004;23(2):638–47.Goodman CM, McCusker CD, Yilmaz T, Rotello VM. Toxicity of gold nanoparticles functionalized with
cationic and anionic side chains. Bioconjug Chem 2004;15(4):897–900.Grinvald A, Lieke E, Frostig RD, Gilbert CD, Wiesel TN. Functional architecture of cortex revealed by
optical imaging of intrinsic signals. Nature 1986;324:361–4.Hacker GW. High performance nanogold-silver in situ hybridisation. Eur J Histochem 1998;42(2):111–20.Hacker GW, Gu J. Gold and silver staining: techniques in molecular morphology. Boca Raton: CRC
Press; 2002.Hacker GW, Tubbs RR. Molecular morphology in human tissues: Techniques and applications. Boca
Raton: CRC Press; 2004.Hacker GW, Graf AH, Thurner J. Application of silver acetate autometallography in histopathology: a
new detection method for use in immunogold silver staining, lectin histochemistry and in situhybridization. Verh Dtsch Ges Pathol 1990;74:368–72.
Hacker GW, Danscher G, Graf AH, Bernatzky G, Schiechl A, Grimelius L. The use of silver acetateautometallography in the detection of catalytic tissue metals and colloidal gold particles bound tomacromolecules. Prog Histochem Cytochem 1991;23(1–4):286–90.
Hacker GW, Graf AH, Hauser-Kronberger C, Wirnsberger G, Schiechl A, Bernatzky G, et al. Applicationof silver acetate autometallography and gold-silver staining methods for in situ DNA hybridization.Chin Med J (Engl) 1993;106(2):83–92.
Hacker GW, Cheung ALM, Tubbs RR, Grimelius L, Danscher G, Hauser-Kronberger C. Immunogold-silver staining for light microscopy using colloidal or clustered gold (NanogoldTM). In: Hacker GW,
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139 133
Gu J, editors. Gold and silver staining: techniques in molecular morphology. Boca Raton: CRC Press;2002. p. 47–69.
Hainfeld JF. A small gold-conjugated antibody label: improved resolution for electron microscopy.Science 1987;236(4800):450–3.
Hainfeld JF. Gold cluster-labelled antibodies. Nature 1988;333(6170):281–2.Hainfeld JF. Labeling with nanogold and undecagold: techniques and results. Scanning Microsc Suppl
1996;10:309–22 (discussion 322-5).Hainfeld JF, Furuya FR. A 1.4-nm gold cluster covalently attached to antibodies improves
immunolabeling. J Histochem Cytochem 1992;40(2):177–84.Hainfeld JF, Powell RD. New frontiers in gold labeling. J Histochem Cytochem 2000;48:471–80.Hainfeld JF, Sprinzl M, Mandiyan V, Tumminia SJ, Boublik M. Localization of a specific nucleotide in
yeast tRNA by scanning transmission electron microscopy using an undecagold cluster. J Struct Biol1991;107(1):1–5.
Hainfeld JF, Powell RD, Stein JK, Hacker GW, Hauser-Kronberger C, Cheung ALM, et al. Gold-basedautometallography. In: Bailey GW, Jerome WG, McKernan S, Mansfield JF, Price RL, editors.Proceedings of the 57th annual meeting, Microscopy Society of America. New York: Springer; 1999.p. 486–7.
Hansen JC, Danscher G. Quantitative and qualitative distribution of mercury in organs from arcticsledgedogs: An atomic absorption spectrophotocmetric and histochemical study of tissue samples fromnatural long-termed high dietary organic mercury-exposed dogs from Thule, Greenland. PharmacolToxicol 1995;77:189–95.
Haug FM. Electron microscopical localization of the zinc in hippocampal mossy fibre synapses by amodified sulfide silver procedure. Histochemie 1967;8(4):355–68.
Haug FM. Heavy metals in the brain. A light microscope study of the rat with Timm’s sulphide silvermethod. Methodological considerations and cytological and regional staining patterns. Adv AnatEmbryol Cell Biol 1973;47:1–71.
Haug FM. Light microscopical mapping of the hippocampal region, the pyriform cortex and thecorticomedial amygdaloid nuclei of the rat with Timm’s sulphide silver method. I. Area dentata,hippocampus and subiculum. Z Anat Entwicklungsgesch 1974;145(1):1–27.
Helling HB. Histochemischer Nachweissdes silbers bei weissen Ratten. Thesis. Gottingen; 1967.Holgate CS, Jackson P, Cowen PN, Bird CC. Immunogold-silver staining: new method of immunostaining
with enhanced sensitivity. J Histochem Cytochem 1983;31:938–44.Holm IE, Andreasen A, Danscher G, Perez-Clausell J, Nielsen H. Quantification of vesicular zinc in the
rat brain. Histochemistry 1988;89(3):289–93.Holm IE, Andreasen A, Danscher G, Nielsen H. Densitometric analysis of the local bleaching of the Neo-
Timm staining pattern following intrahippocampal injection of diethyldithiocarbamate. HistochemJ 1991;23:63–8.
Hørsted-Bindslev P, Bolewska JE, Arenholt-Bindslev D, Danscher G. Dentinal transport of mercury fromdental silver amalgam restorations. Progr Histochem Cytochem 1991;23:321–6.
Howell GA, Frederickson CJ. A retrograde transport method for mapping zinc-containing fiber systems inthe brain. Brain Res 1990;515(1–2):277–86.
Howell GA, Welch MG, Frederickson CJ. Stimulation-induced uptake and release of zinc in hippocampalslices. Nature 1984;308(5961):736–8.
Hurtley SM, Helmuth L. Special issue on biological imaging. Science 2003;300.Ishihara H, Maechler P, Gjinovci A, Herrera PL & Wollheim CB. Islet beta-cell secretion determines
glucagon release from neighbouring alpha-cells. Nat Cell Biol 2003;5:330–5.Jacobsen E, Andreasen A, Graudal H, Danscher G. Autometallographic demonstration of gold uptake in
cultured synovial fluid cells from patients with rheumatoid arthritis. Scand J Rheumatol 1989;18:161–4.
Jo SM, Danscher G, Schrøder HD, Won MH, Cole TB. Zinc-enriched (ZEN) terminals in mouse spinalcord: immunohistochemistry and autometallography. Brain Res 2000;870(1–2):163–9.
Jones A, Moeremans M. Coillidal gold for the detection of proteins on blots and immunoblots. In: WalkerJ, editor. Methods in molecular biology, Vol. 3. Clifton NJ: Humana Press; 1988. p. 441–79.
Josephson L, Tung CH, Moore A, Weissleder R. High-efficiency intracellular magnetic labeling with novelsuperparamagnetic-Tat peptide conjugates. Bioconjug Chem 1999;10(2):186–91.
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139134
Kennedy C, Des Rosiers MH, Sakurada O, Shinohara M, Reivich M, Jehle JW, et al. Metabolic mappingof the primary visual system of the monkey by means of the autoradiographic [14C]deoxyglucosetechnique. Proc Natl Acad Sci USA 1976;73:4230–4.
Keynes RD. The leakage of radioactive potassium from stimulated nerve. J Physiol 1951;113:99–114.Keynes RD, Ritchie JM. The movements of labelled ions in mammalian non-myelinated nerve fibres.
J Physiol 1965;179:333–67.Kim BJ, Kim YH, Kim S, Kim JW, Koh JY, Oh SH, et al. Zinc as a paracrine effector in pancreatic islet
ultrastructural monitoring of zinc in the endocrine pancreas. Histochem Cell Biol 2001;115:125–9.Lah JJ, Hayes DM, Burry RW. A neutral pH silver development method for the visualization of
1-nanometer gold particles in pre-embedding electron microscopic immunocytochemistry. J HistochemCytochem 1990;38(4):503–8 [Erratum in: J Histochem Cytochem 1990;38(9):1396].
Landowne DA. Comparison of radioactive thallium and potassium fluxes in the giant axon of the squid.J Physiol 1975;252:79–96.
Lansdown AB. Silver. I: Its antibacterial properties and mechanism of action. J Wound Care2002;11(4):125–30.
Lansdown AB. A review of the use of silver in wound care: facts and fallacies. Br J Nurs 2004;13(6Suppl):S6–S19.
Larsen M, Bjarkam CR, Stoltenberg M, Sørensen JC, Danscher G. An autometallographic technique formyelin staining in formaldehyde-fixed tissues. Histol Histopathol 2003;18:1125–30.
Laube B. Potentiation of inhibitory glycinergic neurotransmission by Zn2+: a synergistic interplaybetween presynaptic P2� 2 and postsynaptic glycine receptors. Eur J Neurosci 2002;16(6):1025–36.
Lewin M, Carlesso N, Tung CH, Tang XW, Cory D, Scadden DT, et al. Tat peptide-derivatized magneticnanoparticles allow in vivo tracking and recovery of progenitor cells. Nat Biotechnol 2000;18(4):410–4.
Li Y, Hough CJ, Frederickson CJ, Sarvey JM. Induction of mossy fiber – Ca3 long-term potentiationrequires translocation of synaptically released Zn2+. J Neurosci 2001;21(20):8015–25.
Liesegang RE. Die Kolloidchemie der histologischen Silberfarbungen. In: Ostwald W, editor.Kolloidchemische Beihefte (Erganzungshefte zur Kolloid-Zeifschrift). Dresden-Leipzig: Verlag vonTheodor Steinkopff; 1911. p. 1–44.
Liesegang RE, Rieder W. Versuche mit einer ‘‘Keimmethode’’ zum Nachweis von Silber inGewebsschnitten. Z Wiss Mikrosk 1921;38:334–8.
Liuzzi JP, Cousins RJ. Mammalian zinc transporters. Annu Rev Nutr 2004;24:151–72.Long Y, Frederickson CJ. A zinc-containing fiber system of thalamic origin. Neuroreport 1994;
5(16):2026–8.Long Y, Hardwick AL, Frederickson CJ. Zinc-containing innervation of the subicular region in the rat.
Neurochem Int 1995;27(1):95–103.Lormee P, Lecolle S, Septier D, le Denmat D, Goldberg M. Autometallography for histochemical
visualization of rat incisor polyanions with cuprolinic blue. J Histochem Cytochem 1989;37:203–8.Luppo-Cramer. Neue Untersuchungen zur Theorie der phtotgraphischen Vorgange. Photographische
Korrespondenz 1914;640:28–34.Magnusson NE, Larsen A, Rungby J, Kruhoffer M, Orntoft TF, Stoltenberg M. Gene expression changes
induced by bismuth in a macrophage cell line. Cell Tissue Res 2005;321(2):195–210.McPartlin M, Mason R, Malatesta L. Novel cluster complexes of gold(0)–gold(1). J Chem Soc Chem
Commun 1969;334.Mehta AC, Dawson-Butterworth K. Argyria. Electron microscopic study of a case. Br J Dermatol
1966;78:175–9.Miro-Bernie N, Sancho-Bielsa FJ, Lopez-Garcia C, Perez-Clausell J. Retrograde transport of sodium
selenite and intracellular injection of micro-ruby: a combined method to describe the morphology ofzinc-rich neurones. J Neurosci Methods 2003;127(2):199–209.
Moeremans M, Daneels G, Van Dijck A, Langanger G, De May J. Sensitive visualization of antigen-antibody reactions in dot and blot immune overlay assays with immunogold and immunogold/silverstaining. J Immmunol Methods 1984;74:353.
Moeremans M, Daneels G, De Mey J. Sensitive colloidal metal (gold or silver) staining of protein blots onnitrocellulose membranes. Anal Biochem 1985;145(2):315–21.
Møller-Madsen B. Localization of mercury in CNS of the rat. II. Intraperitoneal injection ofmethylmercuric chloride (CH3HgCl) and mercuric chloride (HgCl2). Toxicol Appl Pharmacol1990;103:303–23.
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139 135
Møller-Madsen B. Localization of mercury in CNS of the rat. An autometallographic study. PharmacolToxicol 1994;75(Suppl 1):1–41.
Møller-Madsen B, Danscher G. Transplacental transport of gold in rats exposed to sodiumaurothiomalate. Exp Mol Pathol 1983;39:327–31.
Møller-Madsen B, Danscher G. Localization of mercury in CNS of the rat. IV. The effect of selenium onorally administrated organic and inorganic mercury. Toxicol Appl Pharmacol 1991;108:457–73.
Møller-Madsen B, Thorlacius-Ussing O. Localization of gold in the anterior pituitary gland of ratsexposed to sodiumaurothiomalate. Exp Mol Pathol 1984;41(1):74–80.
Møller-Madsen B, Mogensen SC, Danscher G. Ultrastructural localization of gold in macrophages andmast cells exposed to aurothioglucose. Exp Mol Path 1984;40:148–54.
Møller-Madsen B, Mogensen SC, Danscher G, Rungby J, Thorlacius-Ussing O, Graudal H. Mouseperitoneal cells exposed to sodium aurothiomalate in vivo. Int J Tiss Rac 1985;7:439–42.
Møller-Madsen B, Danscher G, Uldbjerg N, Allen JG. Autometallographic demonstration of gold inhuman fetal liver and placenta. Rheumatol Int 1987;7:47–8.
Montagnese CM, Geneser FA, Krebs JR. Histochemical distribution of zinc in the brain of the zebra finch(Taenopygia guttata). Anat Embryol (Berl) 1993;188(2):173–87.
Nitzan YB, Sekler I, Silverman WF. Histochemical and histofluorescence tracing of chelatable zinc in thedeveloping mouse. Histochem Cytochem 2004;52(4):529–39.
Nørgaard JOR, Møller-Madsen B, Hertel N, Danscher G. Silver enhancement of tissue mercury:demonstration of mercury in autometallographic silver grains from rat kidneys. J HistochemCytochem 1989;37(10):1545–7.
Nørgaard JOR, Møller-Madsen B, Danscher G. Autometallographic localization of mercury in rat kidney– interaction between mercury and selenium. Prog Histochem Cytochem 1991;23(1–4):187–93.
Norseth T, Brendeford M. Intracellular distribution of inorganic and organic mercury in rat liver afterexposure to methylmercury salts. Biochem Pharmacol 1971;20:1101–7.
O’Toole D, Castle LE, Raisbeck MF. Comparison of histochemical autometallography (Danscher’s stain)to chemical analysis for detection of selenium in tissues. Vet Diagn Invest 1995;7(2):281–4.
Palmiter RD, Huang L. Efflux and compartmentalization of zinc by members of the SLC30 family ofsolute carriers. Pflugers Arch 2004;447(5):744–51.
Pamphlett R, Danscher G, Rungby J, Stoltenberg M. Tissue uptake of bismuth from shotgun pellets.Environ Res A 2000a;82:258–62.
Pamphlett R, Stoltenberg M, Rungby J, Danscher G. Uptake of bismuth in motor neurons of mice aftersingle oral doses of bismuth compounds. Neurotoxicol Teratol 2000b;22:559–63.
Patel N, Rocks BF, Bailey MP. A silver enhanced, gold labelled, immunosorbent assay for detectingantibodies to rubella virus. J Clin Pathol 1991;44:334–8.
Patel N, Rocks BF, Iversen SA. Direct measurement of low density lipoprotein in whole blood by silver-enhanced gold-labelled immunoassay. Ann Clin Biochem 1992;29(Part 3):283–6.
Pedersen LH, Stoltenberg M, Ernst E, West MJ. Leydig cell death in rats exposed to bismuth subnitrate.J Appl Toxicol 2003;23(4):235–8.
Perez-Clausell J. Organization of zinc-containing terminal fields in the brain of the lizard Podarcishispanica: a histochemical study. J Comp Neurol 1988;267(2):153–71.
Perez-Clausell J, Danscher G. Intravesicular localization of zinc in rat telencephalic boutons.A histochemical study. Brain Res 1985;337(1):91–8.
Perez-Clausell J, Danscher G. Release of zinc sulphide accumulations into synaptic clefts after in vivoinjection of sodium sulphide. Brain Res 1986;362:358–61.
Pohl K, Stierhof YD. Action of gold chloride (‘‘gold toning’’) on silver-enhanced 1 nm gold markers.Microsc Res Tech 1998;42(1):59–65.
Powell RD, Hainfeld JF. Combined Fluorescent and gold probes for microscopic and morphologicalinvestigations. In: Hacker GW, Gu J, editors. Gold and silver staining: techniques in molecularmorphology. Boca Ratun: CRC Press; 2002.
Querido A. Gold intoxication of nervous elements. On the permeabiligy of the blood-brain-barrier. ActaPsychiatr 1947;12:151.
Ranaldi G, Perozzi G, Truong-Tran A, Zalewski P, Murgia C. Intracellular distribution of labile Zn(II)and zinc transporter expression in kidney and MDCK cells. Am J Physiol Renal Physiol2002;283(6):F1365–75.
Rau R. Have traditional DMARDs had their day? Effectiveness of parenteral gold compared to biologicagents. Clin Rheumatol 2005;24(3):189–202.
Roberts WJ. A new procedure for the detection of gold in animal tissues. Proc R Acad 1935;38:540–4.
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139136
Rocks BF, Bertram VM, Bailey MP. Detection of antibodies to the human immunodeficiency virus by asilver-enhanced gold-labelled immunosorbent assay. Ann Clin Biochem 1990;27(Part 2):114–20.
Rocks BF, Patel N, Bailey MP. Use of a silver-enhanced gold-labelled immunoassay for detection ofantibodies to the human immunodeficiency virus in whole blood samples. Ann Clin Biochem1991;28(Part 2):155–9.
Rohringer R, Holden DW. Protein blotting: detection of proteins with colloidal gold, and of glycoproteinsand lectins with biotin-conjugated and enzyme probes. Anal Biochem 1985;144(1):118–27.
Ross JF, Broadwell RD, Poston MR, Lawhorn GT. Highest brain bismuth levels and neuropathology areadjacent to fenestrated blood vessels in mouse brain after intraperitoneal dosing of bismuth subnitrate.Toxicol Appl Pharmacol 1994;124:191–200.
Ross JF, Switzer RC, Poston MR, Lawhorn GT. Distribution of bismuth in the brain after intraperitonealdosing of bismuth subnitrate in mice: implications for routes of entry of xenobiotic metals into thebrain. Brain Res 1996;725:137–54.
Roth J. Applications of immunocolloids in light microscopy. Preparation of protein A-silver and proteinA–gold complexes and their application for localization of single and multiple antigens in paraffinsections. J Histochem Cytochem 1982a;30(7):691–6.
Roth J. The preparation of protein A–gold complexes with 3 and 15 nm gold particles and their use inlabelling multiple antigens on ultra-thin sections. Histochem J 1982b;14(5):791–801.
Rubio ME, Juiz JM. Chemical anatomy of excitatory endings in the dorsal cochlear nucleus of the rat:differential synaptic distribution of aspartate aminotransferase, glutamate, and vesicular zinc. J CompNeurol 1998;399(3):341–58.
Rungby J. Silver-induced lipid peroxidation in mice: interactions with selenium and nickel. Toxicology1987;45(2):135–42.
Rungby J. An experimental study on silver in the nervous system and on aspects of its general cellulartoxicity. Dan Med Bull 1990;37(5):442–9.
Rungby J, Ellermann-Eriksen S, Danscher G. Effects of selenium on toxicity and ultrastructurallocalization of silver in cultured macrophages. Arch Toxicol 1987;61(1):40–5.
Rungby J, Danscher G, Christensen M, Ellermann-Eriksen S, Mogensen SC. Autometallography used as ahistochemical indicator of lysosome function in cultured cells. Histochemistry 1990;94(1):109–11.
Schiønning JD. Retrograde axonal transport of mercury in rat sciatic nerve. Toxicol Appl Pharmacol1993;121:43–9.
Schiønning JD. Experimental neurotoxicity of mercury. Autometallographic and stereologic studies on ratdorsal root ganglion and spinal cord. APMIS 2000;108(Suppl 99):1–32.
Schiønning J, Møller-Madsen B. Autometallographic mapping of mercury deposits in the spinal cord ofrats treated with inorganic mercury. Acta Neuropathol 1991;81:434–42.
Schiønning JD, Danscher G. Autometallographic mercury correlates with degenerative changes in dorsalroot ganglia of rats intoxicated with organic mercury. APMIS 1999;107:303–10.
Schiønning JD, Møller-Madsen B. Autometallographic detection of mercury in rat spinal cord aftertreatment with organic mercury. Virchows Arch B 1992;61:307–13.
Schiønning JD, Poulsen EH, Møller-Madsen B, Danscher G. Autometallographic detection of gold indorsal root ganglia of rats treated with sodium aurothiomalate. Exp Mol Pathol 1992;56:239–47.
Schiønning JD, Danscher G, Christensen MM, Ernst E, Møller-Madsen B. Differentiation of silver-enhanced mercury and gold in tissue sections. J Histochem 1993;25:107–11.
Schiønning JD, Larsen JO, Tandrup T, Braendgaard H. Selective degeneration of dorsal root ganglia anddorsal nerve roots in methyl mercury-intoxicated rats: a stereological study. Acta Neuropathol (Berl)1998;96(2):191–201.
Schrøder HD, Danscher G, Jo SM, Su H. Zinc-enriched boutons in rat spinal cord. Brain Res2000;868(1):119–22.
Scopsi L, Larsson LI. Colloidal gold probes in immunocytochemistry. An optimization of theirapplication in light microscopy by use of silver intensification procedures. Med Biol 1986;64(2–3):139–45.
Seress L, Gallyas F. The use of a sodium tungstate developer markedly improves the electron microscopiclocalization of zinc by the Timm method. J Neurosci Methods 2000;100(1–2):33–9.
Shirabe T. Electron microscopic X-ray microanalysis of the nervous system after mercury intoxication.Folia Psychiatr Neurol Japan 1978;32:278–83.
Shirabe T. Identification of mercury in the brain of Minamata disease victims by electron microscopicX-ray microanalysis. Neurotoxicology 1979;1:349–56.
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139 137
Skutelsky E, Roth J. Cationic colloidal gold–a new probe for the detection of anionic cell surface sites byelectron microscopy. J Histochem Cytochem 1986;34(5):693–6.
Skutelsky E, Goyal V, Alroy J. The use of avidin–gold complex for light microscopic localization of lectinreceptors. Histochemistry 1987;86(3):291–5.
Sliwa-Tomczok W, Tomczok J. Electron microscopical localization of the lead in peripheral bloodneutrophils of the rat with Timm sulphide silver method and X-ray probe microanalysis. Z MikroskAnat Forsch 1990;104(3):458–64.
Slomianka L. Neurons of origin of zinc-containing pathways and the distribution of zinc-containingboutons in the hippocampal region of the rat. Neuroscience 1992;48(2):325–52.
Sloviter RS. A simplified Timm stain procedure compatible with formaldehyde fixation and routineparaffin embedding of rat brain. Brain Res Bull 1990;8:771–4.
Søndergaard LG, Stoltenberg M, Flyvbjerg A, Brock B, Schmitz O, Danscher G, et al. Zinc ions in beta-cells of obese, insulin-resistant, and type 2 diabetic rats traced by autometallography. APMIS2003;111(12):1147–54.
Søndergaard LG, Brock B, Stoltenberg M, Flyvbjerg A, Schmitz O, Smidt K, et al. Zinc fluxes duringacute and chronic exposure of INS-1E cells to increasing glucose levels. Horm Metab Res2005;37(3):133–9.
Sørensen MB, Stoltenberg M, Juhl S, Danscher G, Ernst E. Ultrastructural localization of zinc ions in therat prostate: an autometallographic study. Prostate 1997;31:125–30.
Sørensen MB, Stoltenberg M, Henriksen K, Ernst E, Danscher G, Parvinen M. Histochemical tracing ofzinc ions in the rat testis. Mol Hum Reprod 1998;4:423–8.
Sørensen MB, Bergdahl IA, Hjøllund NHI, Bonde JPE, Stoltenberg M, Ernst E. Zinc, magnesium, andcalcium in human seminal fluid: Relations to other semen parameters and fertility. Mol Hum Reprod1999;5:331–7.
Sørensen FW, Larsen JO, Eide R, Schiønning JD. Neuron loss in cerebellar cortex of rats exposed tomercury vapor: A stereological study. Acta Neuropathol 2000;100:95–100.
Soto M, Cajaraville MP, Marigomez I. Tissue and cell distribution of copper, zinc and cadmium in themussel, Mytilus galloprovincialis, determined by autometallography. Tissue Cell 1996;28:557–68.
Stierhof YD, Humbel BM, Schwarz H. Suitability of different silver enhancement methods applied to 1 nmcolloidal gold particles: an immunoelectron microscopic study. J Electron Microsc Tech1991;17(3):336–43.
Stoltenberg M. Bismuth. Some aspects of localization, transport and pathological effects of metallicbismuth and bismuth salts with special emphasis on its neurotoxicity to man and experimental animals.Doctoral thesis. University of Aarhus; 2004.
Stoltenberg M, Danscher G. Histochemical differentiation of autometallographically traceable metals(Au, Ag, Hg, Bi, Zn). Protocols for chemical removal of separate autometallographic metal clusters inEpon sections. Histochem J 2000;32:645–52.
Stoltenberg M, Hutson JC. Bismuth uptake in rat testicular macrophages. A follow up observationsuggesting that bismuth alters interactions between testicular macrophages and Leydig cells.J Histochem Cytochem 2004;52(9):1241–3.
Stoltenberg M, Ernst E, Andreasen A, Danscher G. Histochemical localization of zinc ions in theepididymis of the rat. Histochem J 1996;28:173–85.
Stoltenberg M, Sørensen MB, Danscher G. Histochemical demonstration of zinc ions in ejaculated humansemen. Int J Androl 1997a;20:229–36.
Stoltenberg M, Lund L, Juhl S, Danscher G, Ernst E. Histochemical demonstration of zinc ions in humanepididymis using autometallography. Histochem J 1997b;29:721–6.
Stoltenberg M, Danscher G, Pamphlett R, Christensen MM, Rungby J. Histochemical tracing of bismuthin testis from rats exposed intraperitoneally to bismuth subnitrate. Reprod Toxicol 2000;14:65–71.
Stoltenberg M, Schiønning JD, Danscher G. Retrograde axonal transport of bismuth. An autometallo-graphic study. Acta Neurophatol 2001a;101:123–8.
Stoltenberg M, Hogenhuis JA, Hauw JJ, Danscher G. Autometallographic tracing of bismuth in humanbrain autopsies. J Neuropathol Exp Neurol 2001b;60:705–10.
Stoltenberg M, Martiny M, Sørensen K, Rungby J, Krogfelt KA. Histochemical tracing of bismuth inHelicobacter pylori after in vitro exposure to bismuth citrate. Scand J Gastroenterol 2001c;2:144–8.
Stoltenberg M, Flyvbjerg A, Søndergaard LG, Rungby J. Decreased serum testosterone levels in ratsexposed intraperitoneally to bismuth subnitrate. J Appl Toxicol 2002a;22:111–5.
Stoltenberg M, Larsen A, Zhao M, Svensson, Danscher G, Brunk U. Bismuth induced lysosomalinstability in J-774 cells. APMIS 2002b;110:396–402.
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139138
Stoltenberg M, Locht L, Larsen A, Jensen D, Danscher G. In vivo cellular uptake of bismuth ions fromshotgun pellets. Histol Histopathol 2003a;18:781–5.
Stoltenberg M, Schiønning JD, West MJ, Danscher G. Bismuth-induced neuronal cell death in rat dorsalroot ganglion: a stereological study. Acta Neuropathol 2003b;105:351–7.
Suh SW, Jensen KB, Jensen MS, Silva DS, Kesslak PJ, Danscher G, et al. Histochemically-reactive zinc inamyloid plaques, angiopathy, and degenerating neurons of Alzheimer’s diseased brains. Brain Res2000;852:274–8.
Tauck DL, Nadler JV. Evidence of functional mossy fiber sprouting in hippocampal formation of kainicacid-treated rats. J Neurosci 1985;5:1016–22.
Thorlacius-Ussing O, Møller-Madsen B, Danscher G. Intracellular accumulation of mercury in theanterior pituitary of rats exposed to mercuric chloride. Exp Mol Pathol 1985;42:278–86.
Timm F. Zur Histochemie der Schwermetalle. Das Sulfid-Silberverfahren. Dtsch Z Gerichtl Med1958;46:706–11.
Timm F. Der histochemische nachweis des kupfers im gehirn. Histochemie 1961;2:332–41.Timm F. Histochemische Lokalisation und Nachweis der Schwermetalle. Acta Histochem 1962(Suppl.
3):142–58.Traber KE, Okamoto H, Kurono C, Baba M, Saliou C, Soji T, et al. Anti-rheumatic compound
aurothioglucose inhibits tumor necrosis factor-alpha-induced HIV-1 replication in latently infectedOM10.1 and Ach2 cells. Int Immunol 1999;11(2):143–50.
Tubbs R, Pettay J, Skacel M, Powell R, Stoler M, Roche P, et al. Gold-facilitated in situ hybridization: abright-field autometallographic alternative to fluorescence in situ hybridization for detection of Her-2/neu gene amplification. Am J Pathol 2002;160(5):1589–95.
Ueno S, Tsukamoto M, Hirano T, Kikuchi K, Yamada MK, Nishiyama N, et al. Mossy fiber Zn2+spillover modulates heterosynaptic N-methyl-D-aspartate receptor activity in hippocampal CA3circuits. J Cell Biol 2002;158(2):215–20.
Valente T, Auladell C, Perez-Clausell J. Postnatal development of zinc-rich terminal fields in the brain ofthe rat. Exp Neurol 2002;174(2):215–29.
Voigt GE. Unterschungen mit der Sulfidsilbermethode an menschlichen und tierischen Bauchspeichel-drusen. Virchows Arch Path Anat 1959;332:295–323.
Wang Z, Danscher G, Jo SM, Shi Y, Schrøder HD. Retrograde tracing of zinc-enriched (ZEN) neuronalsomata in rat spinal cord. Brain Res 2001a;900(1):80–7.
Wang Z, Li JY, Dahlstrom A, Danscher G. Zinc-enriched GABAergic terminals in mouse spinal cord.Brain Res 2001b;921(1–2):165–72.
Wang Z, Danscher G, Kim YK, Dahlstrom A, Jo SM. Inhibitory zinc-enriched terminals in the mousecerebellum: double-immunohistochemistry for zinc transporter 3 and glutamate decarboxylase.Neurosci Lett 2002a;321(1–2):37–40.
Wang ZY, Li JY, Danscher G, Dahlstrom A. Localization of zinc-enriched neurons in the mouseperipheral sympathetic system. Brain Res 2002b;928(1–2):165–74.
Wang ZY, Danscher G, Dahlstrom A, Li JY. Zinc transporter 3 and zinc ions in the rodent superiorcervical ganglion neurons. Neuroscience 2003;120(3):605–16.
Wang ZY, Stoltenberg M, Huang L, Danscher G, Dahlstrom A, Shi Y, et al. Abundant expression of zinctransporters in Bergman glia of mouse cerebellum. Brain Res Bull 2005;64(5):441–8.
Weiss JH, Sensi SL, Koh JY. Zn(2+): a novel ionic mediator of neural injury in brain disease. TrendsPharmacol Sci 2000;21(10):395–401.
Wen GY, Wisniewski HM. Histochemical localization of aluminum in the rabbit CNS. Acta NeuropatholBerl 1985;68:175–84.
Wenzel HJ, Cole TB, Born DE, Schwartzkroin PA, Palmiter RD. Ultrastructural localization of zinctransporter-3 (ZnT-3) to synaptic vesicle membranes within mossy fiber boutons in the hippocampus ofmouse and monkey. Proc Natl Acad Sci USA 1997;94:12676–81.
Wu X, Liu H, Liu J, Haley KN, Treadway JA, Larson JP, et al. Immunofluorescent labeling of cancermarker Her2 and other cellular targets with semiconductor quantum dots. Nat Biotechnol2003;21(1):41–6.
Xiao Y, Patolsky F, Katz E, Hainfeld JF, Willner I. ‘‘Plugging into Enzymes’’: nanowiring of redoxenzymes by a gold nanoparticle. Science 2003;299(5614):1877–81.
Yang JP, Merin JP, Nakano T, Kato T, Kitade Y, Okamoto T. Inhibition of the DNA-binding activity ofNF-kappa B by gold compounds in vitro. FEBS Lett 1995;361(1):89–96.
Yoshida S, Kato T, Sakurada S, Kurono C, Yang JP, Matsui N, et al. Inhibition of IL-6 and IL-8induction from cultured rheumatoid synovial fibroblasts by treatment with aurothioglucose. IntImmunol 1999;11(2):151–8.
ARTICLE IN PRESS
G. Danscher, M. Stoltenberg / Progress in Histochemistry and Cytochemistry 41 (2006) 57–139 139
Yuan XM. Apoptotic macrophage-derived foam cells of human atheromas are rich in iron andferritin, suggesting iron-catalysed reactions to be involved in apoptosis. Free Radic Res 1999;30:221–31.
Zdolsek JM, Roberg K, Brunk UT. Visualization of iron in cultured macrophages: a cytochemical lightand electron microscopic study using autometallography. Free Radic Biol Med 1993;15:1–11.
Zeiger K. Physikochemische Grundlagen der histologischen Methodik. Wiss Forschungsber 1938;48:55–105.
