Gold Toning Research Articles

Valence quark distribution functions of pion and kaon in the modified chiral quark model

We investigate the structure of pion and kaon based on the modified chiral quark model (χQM). To this end, we calculate the valence quark distribution functions of pion and kaon using the modified χQM in which the bare quarks inside these mesons are dressed by the clouds of Goldstone (GS) bosons and gluons. In order to compute the valence distributions of the light mesons using the modified χQM, the bare quark distribution functions of these mesons are needed. We apply the bare distributions which have been obtained via a phenomenological approach. We compare the results of our theoretical model for the valence distributions of the light mesons with those of some parametrization models and also the available experimental data.

Pioneering a golden age of cerebral microcircuits: The births of the combined Golgi–electron microscope methods

Theodor W. Blackstad devised methods by which the synaptic connectivity of neuron somata and their dendritic and axonal processes in the CNS could be analyzed by the combined use of light and electron microscope techniques. His first publication on that subject dates from 1965 and was contemporary to the independent research by William K. Stell. The Golgi method was an obvious neuronal marker at those times, and Blackstad and Stell showed that the Golgi precipitate is electron-dense and intracellular and, therefore, it could help identify in the electron microscope, with great accuracy, profiles of neurons initially visualized in light microscopy. Besides this convergent research, Blackstad demonstrated for the first time that anterograde axonal degeneration could be combined with the Golgi–electron microscope method, allowing the identification of the neurons whose dendritic or somatic profiles were postsynaptic to the severed axonal afferent projections. Last, but not least, Blackstad pioneered de-impregnation techniques for electron microscopy of Golgi preparations. This had a great impact in the study of synaptic circuitry. The present account is a remembrance of the events that linked these early attempts with the development of a de-impregnation method based on gold toning by Alan Peters and the present author.

Examining neocortical circuits: some background and facts.

The first definitive studies of where afferents to cerebral cortex terminate were made possible by the finding that as they degenerate axon terminals become electron dense. Gold toning of Golgi impregnated neurons allowed the postsynaptic targets of these afferents to be identified by electron microscopy and also allowed the termination sites of axons from a variety of types of cortical neurons to be ascertained, while the development of antibodies to GAD and to GABA made it possible to determine which types of cortical neurons are inhibitory. Subsequently the use of gold toned, Golgi impregnated material to examine neuronal connectivity was made redundant by the development of techniques that allowed the physiological properties of cortical neurons to be evaluated in neurons filled intracellularly with markers. Intracellular filling showed the axonal trees of cortical neurons are much more widespread than had been revealed by Golgi impregnations. As a result of numerous studies of the axons of identified neurons, we know a great deal about where most of the different types of neurons in cerebral cortex form their synapses, but on the other side of the picture there is a dearth of information about the origins of the inputs that specific types of cortical neurons receive. However, it is evident that each cortical neuron is the focus of input from many other neurons, and on the basis of the available data it is estimated that a single pyramidal cell in cortex receives its input from as many as 1,000 other excitatory neurons and as many as 75 inhibitory neurons.

A practical technique to postfix nanogold-immunolabeled specimens with osmium and to embed them in Epon for electron microscopy.

Nanogold is a tiny gold probe, freely diffusible in cells and tissues, and is suitable for pre-embedding immunohistochemistry. However, it is necessary to develop Nanogold to a larger size so that it can be observed by conventional transmission electron microscopy. Silver enhancement is usually used for visualizing Nanogold, but the silver shell produced is unstable in OsO(4) and often becomes invisible after OsO(4) postfixation, which is necessary for good visualization of ultrastructure. We used silver enhancement with silver acetate, followed by gold toning with chloroauric acid, to replace the silver shell with a more stable gold in order to observe Nanogold after osmium fixation and Epon embedding. This technique is applicable to various intra- and extracellular antigens. For correlative observation of immunolabled specimens by light and electron microscopy, specimens adhered to slideglasses were embedded in Epon under non-adhesive plastic film. By heating the Epon sheets after polymerization, these supports were removed without difficulty and provided easy correlative observation.

Gold toning preserves integrity of silver enhanced immunogold particles during osmium tetroxide treatment for demonstration of a biogenic amine

We describe a protocol that enhances immunolabelling of nervous tissue for ultrastructural study. Insect tissue is fixed, sectioned, and labelled with a polyclonal antiserum against serotonin and a secondary antibody conjugated with 1 nm colloidal gold. The gold particles are silver-enhanced to ease detection and then protected by gold toning. Finally, the tissue is post fixed in glutaraldehyde fixative followed by osmium tetroxide and further processed for electron microscopy. We demonstrated on insect nervous tissue that gold toning protects marker particles from the influence of osmium tetroxide. Use of buffered solutions throughout the protocol led to well preserved ultrastructural details, and marker particle size was not reduced with a short gold toning time. We also suggest use of this protocol for vertebrate or other invertebrate tissue. Theme B: Cellular and Molecular Biology Topic 24: Staining Tracing and Imaging Techniques

Action of gold chloride (“gold toning”) on silver‐enhanced 1 nm gold markers

In conventional immunoelectron microscopy (IEM), very small colloidal gold particles (0.8–3 nm), or the gold compound Nanogold (1.4 nm) are silver-enhanced for easy detection. However, silver enhancement has drawbacks. First, the silver layer is dissolved during fixation with osmium tetroxide, even if the concentration and incubation time are strongly reduced during pre-embedding labeling experiments in transmission electron microscopic (TEM) and scanning electron microscopic (SEM) studies. Second, after exposure to the electron beam the silver layer may migrate on the section or the whole particles may disappear. Sometimes silver migration can be observed even without irradiation. This effect strongly hampers reinvestigation of previously inspected areas, after some time of storage. In both cases, gold chloride treatment after silver enhancement is sufficient to completely protect the silver-enhanced 1 nm gold markers. Gold chloride treatment is part of the so-called “gold toning” procedure, which is a method used to substitute and/or cover the silver by a layer of gold. It can be applied in TEM and SEM experiments. As a serious drawback, gold chloride treatment slightly reduces the size of both unenhanced and silver-enhanced gold particles and can lead to disintegrated silver/gold particles. Therefore, this technique is useful for pre-embedding IEM, on-(resin)section, and ultrathin cryosection labeling experiments. However, it appears to be unsuitable for double-labeling studies using different gold sizes, for quantitation experiments, and in SEM. Microsc. Res. Tech. 42:59–65, 1998. © 1998 Wiley-Liss, Inc.

Preparation, use, and enlargement of ultrasmall gold particles in immunoelectron microscopy.

The introduction of ultrasmall (approximately 1-3 nm) colloidal gold markers in immunoelectron microscopy (IEM) in 1989 has considerably improved the sensitivity of this marker system. Ultrasmall gold markers have opened the field of pre-embedding labeling studies to gold markers without the need of harsh permeabilizing steps. They are recommended for the detection of scarce antigens in ultrathin cryosections which may otherwise escape immunodetection. However, reports concerning the preparation of ultrasmall gold colloids, their conjugation to proteins, and their use in high-resolution studies (without an additional enlargement step) are very limited. Also, the available enlargement techniques necessary for the use of this marker in conventional electron microscopy require detailed discussion to clarify the large number of contradictory observations. The present review summarizes and discusses the findings accumulated within the last 10 years on the application of ultrasmall gold markers in IEM with regard to their merits, limitations, detection sensitivity, and suitability for different labeling techniques. It should provide practical hints for the use of ultrasmall gold colloids and discusses problems arising with enlargement techniques such as silver enhancement and gold toning procedures.

Gold Toning for Silver Enhanced Immunogold Reacted Tissue:

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Immunocytochemistry of extracellular matrix components in the rat seminiferous tubule: electron microscopic localization with improved methodology.

Cell-cell interactions between Sertoli, myoid, Leydig, and germ cells are thought to be essential for spermatogenesis. These cells interact with each other through the extracellular matrices (ECMs) of the testicular lamina propria, which thus may have an important function in spermatogenesis. For an understanding of the role of ECMs in spermatogenesis, it is important to investigate the molecular constitution of the testicular ECM. We examined the distribution of type IV, V, and VI collagens (Cols), laminin (LM), fibronectin (FN), and heparan sulfate proteoglycan (HSPG) in the rat testicular lamina propria. Adult rat testes were fixed with 4% paraformaldehyde and 0.2% glutaraldehyde. Ultrathin frozen sections were made and immunolabeling was performed according to the method of Tokuyasu (1986 J. Microsc., 143:139-149). Alternatively, for preembedding immunocytochemistry, we used labeling with nanogold, followed by silver enhancement and gold toning. The tissues were then fixed with osmium and embedded in Epon (Sawada and Esaki 1994 J. Electron Microsc., 43:361-366). These specimens were examined in a JEOL JEM-100C transmission electron microscope operated at 80 kV. Col IV, LM, and HSPG were localized in the basement membranes of the seminiferous tubule (ST-BM) and in the BM of myoid cells. Cols V and VI, and FN were localized both in the connective tissue between the seminiferous tubule and the myoid cells (tubule-myoid connective tissue) and in the connective tissue between the myoid cells and the lymphatic endothelial cells (myoid-endothelium connective tissue). Furthermore, statistical analysis of the micrographs of immunogold labeling for Col IV, LM, and FN around the ST-BM suggested that the Col IV molecule is located uniformly in the ST-BM, whereas the LM molecule is distributed mainly in the lamina rara of ST-BM, and the FN molecule appears to be present predominantly in the tubule-myoid connective tissue. Distinct distribution patterns were observed for each antigen within the testicular lamina propria at the ultrastructural level. However, localization of some components was not consistent with localizations reported by others.

The mechanism of Bodian's silver staining: effect of copper ion on silver impregnation

We studied the effect of the duration (0.5–48 h) of silver impregnation on the intensity of Bodian's silver staining using formalin-fixed, paraffin-embedded sections of a human brain. The silver ion (Ag +) and copper ion (Cu 2+) in the silver protein solution were quantified simultaneously for treatments of various durations. Both the intensity of staining and the quantities of Ag + and Cu 2+ were greatly affected by the duration of silver impregnation. While the quantity of Ag + considerably decreased during the first 4 h of impregnation, that of Cu 2+ greatly increased. Only small changes were observed in both ions after 12 h. Neurofibrils or axons, and neurofibrillary tangles (NFTs) were clearly stained after 12–14 and 16–48 h of impregnation, respectively. Strong staining of these components was not observed for other durations of treatment. The amount of metallic copper in silver impregnation also affected both the intensity of staining and the quantities of Ag + and Cu 2+ in the silver protein solution. Ag + and Cu 2+ were also present in the gold trichloride acid solution in which the section was toned. These findings suggest that both Cu 2+ derived from metallic copper and silver protein are deposited on sections during silver impregnation, that the amount of Cu 2+ may determine the amount of silver protein deposited on the section, and that the reduced (metallic) form of silver and copper on the section may participate in gold toning. Thus, to achieve strong staining of a desired component, it is important to examine the conditions of silver impregnation (i.e. duration and amount of metallic copper). For strong staining of neurofibrils, axons and NFTs, optimal results are obtained by the addition of 5 g of metallic copper foil to 100 ml of 1% silver protein solution, and by 16–24 h of impregnation.

Use of Nanogold Followed by Silver Enhancement and Gold Toning for Preembedding Immunolocalization in Osmium-fixed, Epon-embedded Tissues

A reliable, sensitive, high-resolution method with good structural visualization for preembedding immunoelectron microscopy was proposed. The present technique involves the following processes. Immunolabeling of cryostat sections with primary antibodies and with nanogolds, silver intensification for visualization of nanogold secondary antibodies, gold-toning for stabilization of silver shells, and osmium postfixation and embedding in Epon for good structural visualization. The technique was applied to rat testis with anti-laminin and anti-fibronectin antibodies, showing that these two proteins are localized differentially in the lamina propria of the tissue.

Localization of immunoreactive tyrosine hydroxylase in the goldfish retina with pre-embedding immunolabeling with one-nanometer colloidal gold particles and gold toning.

The goal of this study was to develop an alternative to silver intensification for visualizing small colloidal gold particles by light and electron microscopy. The isolated goldfish retina was labeled with rabbit antiserum to tyrosine hydroxylase and 1-nm colloidal gold-conjugated goat anti-rabbit IgG. The gold particles were enlarged by toning with gold chloride, followed by reduction in oxalic acid. Dopaminergic interplexiform cells were clearly visible by light microscopy and, in lightly-fixed material treated with detergent, they were labeled in their entirety. Labeling was qualitatively similar, although less extensive, in material fixed and processed for electron microscopy. The labeled processes were apparent in ultra-thin sections viewed at low magnification, but the gold-toned particles were not so large that they obscured subcellular structures. The procedure apparently had no deleterious effects on the tissue, since the ultrastructural preservation was comparable to that seen with other pre-embedding immunolabeling methods. The technique was simple, reliable and, since the gold solutions were so dilute, relatively inexpensive.

Intensification of labelings of the immunogold silver staining method by gold toning

We evaluated the applicability of the gold toning procedure to the immunogold silver staining method using monoclonal antibody against dopamine. Immunolabeling was examined in the rat substantia nigra at light and electron microscopic levels. Vibratome sections of fixed midbrains were incubated with anti-dopamine antiserum and then with 5 nm colloidal gold bound to goat anti-mouse immunoglobulin. Silver staining of these sections produced a light brown immunolabeling. After the sections were processed by gold toning, the labeling became intense black. At a light microscopic level, these high contrast signals were retained after the sections were osmicated and embedded in Epon. At an electron microscopic level, signal-to-noise ratio was high, and the positive staining could easily be verified at a low-power magnification. The technique described here, the gold-toned immunogold silver staining method, provides high contrast signals and is much more sensitive than immunogold silver staining alone. This method, therefore, has great potential for use in immunohistochemical analysis of the central nervous system.

Gold Toning Improves the Visualization of Nucleolar Organizer Regions in Paraffin Embedded Tissues

A modification of the silver colloid technique for staining nucleoar organizer regions in paraffin embedded tissues is described. This modification involves the application of a gold toning step with subsequent gold reduction, if necessary, following incubation of sections in the standard silver colloid solution. Silver stained nucleolar organizer regions (AgNORs) in toned sections are more sharply delineated when compared to untoned controls. In high grade tumors the addition of the toning step results in significantly higher AgNOR counts due to the ability to discriminate more easily individual AgNORs in argyrophilic aggregates within the nucleus. It is recommended, because of enhanced visualization, that this modification of the silver colloid technique be used in studies involving quantification of AgNORs in tissue sections.

Cholera toxin B‐gold, a retrograde tracer that can be used in light and electron microscopic immunocytochemical studies

The purpose of this study was to test whether a new retrograde tracer, the B subunit of cholera toxin conjugated to colloidal gold particles (CTB-gold), was taken up and transported by neurons in the central nervous system of the rat. Retrograde transport of CTB-gold was assessed from axon terminals, from damaged nerve fibers, and from axons of passage. For light microscopy, CTB-gold was visualized by silver intensification; for electron microscopy, sections were silver-intensified with or without subsequent gold toning. Retrogradely transported CTB-gold was detected in neurons after survival times of 12 hours to 42 days and appeared as black punctate deposits in perikarya and proximal dendrites at the light microscope level. Ultrastructurally, the deposits were usually associated with lysosomes. Injections of CTB-gold into the caudal ventrolateral medulla or into the lateral horn of the spinal cord gave small well-defined injection sites and resulted in retrograde labelling in medullary neurons in the same locations as similarly placed injections of wheat germ agglutinin-horseradish peroxidase. When injected into the superior cervical ganglion, CTB-gold was transported to nerve cell bodies in the spinal cord, but application of CTB-gold to the cut cervical sympathetic trunk did not label neurons in the spinal cord. Injection of CTB-gold into the nodose ganglion retrogradely labelled neurons in the dorsal motor nucleus of the vagus and the nucleus ambiguus. CTB-gold was not transported anterogradely from injections sites within the medulla. Nerve fibers and cell bodies containing neuropeptides, monoamines, or neurotransmitter-synthesizing enzymes were readily immunostained after silver intensification of retrogradely transported CTB-gold. Immunoreactivity for neuropeptides and enzymes was also demonstrated ultrastructurally after silver intensification and gold toning. These results show that CTB-gold is retrogradely transported from nerve terminals and fibers of passage but not from damaged axons. CTB-gold gives well-localized injection sites and persists in neurons for weeks. Transported CTB-gold is easily visualized and its detection is compatible with light and electron microscopic immunocytochemistry. These properties make CTB-gold a valuable tool for studying the connectivity and neurochemistry of pathways in the central nervous system.

Structural alterations of dendritic spines induced by neural degeneration of their presynaptic afferents.

Morphological parameters were compared for dendritic spines of spiny stellate neurons in layer IV of the barrel region of mouse somatosensory cortex, which synapse with degenerated thalamocortical afferents (TC spines) and with intact, unidentified axon terminals (UI spines). Spiny stellate neurons were labeled for light and electron microscopic identification by Golgi impregnation and gold toning. Dendritic spines were examined in series of thin sections, and TC spines were ultrastructurally detectable because of the degeneration-induced characteristic appearance of the TC axon terminals. Results show that the means of the width of the spine head and of the length of the spine stalk were significantly higher in TC spines than in UI spines by about 11 and 25%, respectively. The variability of these two morphological parameters was significantly lower for TC spines. The mean of the spine stalk width at the narrowest cross section of the stalk was about 0.12 microns, with no significant difference observed between the two spine groups. No specific relationship was found in either the TC or the UI groups of spines between the length of the spine stalk and the width of the spine stalk at its narrowest profile. As structural features typifying transneuronal degeneration were not observed along the dendritic spines examined, it is speculated that the morphological differences encountered between the TC and UI spines may result, at least in part, from the degeneration-induced synaptic inactivity of the TC axospinous synapses, rather than exclusively from any direct effects of the degeneration process.

Enigmatic bipolar cell of rat visual cortex

Our earlier Golgi-electron microscopic study of bipolar cells in the rat visual cortex showed the axons of these neurons as forming asymmetric synapses (Peters and Kimerer; J. Neurocytol, 10:921-946, '81) in which the most common postsynaptic elements were dendritic spines. This result was unexpected, since Parnavelas (Parnavelas, Sullivan, Lieberman, and Webster: Cell Tissue Res. 183:499-517, '77) had earlier shown a bipolar cell from the same cortex to have an axon forming symmetric synapses with dendritic shafts. Here then was an enigma, strengthened by examination of neuronal components labelled by antibodies to two compounds in particular--namely, vasoactive intestinal polypeptide (VIP) and choline acetyltransferase (ChAT). Antibodies to these compounds preferentially label bipolar cells in the rat cerebral cortex, and the labelled axon terminals form symmetric synapses. Against this background the present study was performed, and it has been shown that the resolution to the enigma is that there are two different populations of bipolar cells in the rat visual cortex. Thus some Golgi-impregnated bipolar cells examined by electron microscopy after gold toning have been found to possess axons forming asymmetric synapses, and others have been found to have axons forming symmetric synapses. The axons of the bipolar cells forming asymmetric synapses most commonly synapse with dendritic spines (67%), although other terminals synapse with dendritic shafts (33%). In contrast, the bipolar cells with axons forming symmetric synapses preferentially synapse with dendritic shafts (100%). The population of bipolar cells that form symmetric synapses includes the ones that label with antibodies to vasoactive intestinal polypeptide (VIP), for the axons of VIP-labelled bipolar cells have been traced to labelled terminals forming symmetric synapses. However, examination of the population of VIP-labelled axon terminals shows that in addition to dendritic shafts, some of the labelled terminals synapse with the cell bodies of pyramidal and nonpyramidal cells. This includes bipolar cells, some of which receive large numbers of VIP-labelled axon terminals. It is also shown that some VIP-positive bipolar cells have myelinated axons. Analysis of tissue labelled with VIP antibody reveals that about 50% of the total population of bipolar cells in the rat visual cortex is VIP positive. These results are discussed in the light of information about labelling of bipolar cells with antibodies to gamma-aminobutyric acid (GABA) and to other peptides, and it is suggested that most VIP-positive bipolar cells also contain GABA.

Confocal laser microscopy of impregnated central nervous system tissue

A classic preparation of central nervous system tissue (CNS) is the Golgi procedure popularized by Cajal. The method is partially specific as only a few cells are impregnated with silver chromate usualy after osmium post fixation. Samples are observable by light (LM) or electron microscopy (EM). However, the impregnation is often so dense that structures are masked in EM, and the osmium background may be undesirable in LM. Gold toning is used for a subtle but high contrast EM preparation, and osmium can be omitted for LM. We are investigating these preparations as part of a study to develop correlative LM and EM (particularly HVEM) methodologies in neurobiology. Confocal light microscopy is particularly useful as the impregnated cells have extensive three-dimensional structure in tissue samples from one to several hundred micrometers thick. Boyde has observed similar preparations in the tandem scanning reflected light microscope (TSRLM).

Palladium Toning of Silver-Impregnated Reticular Fibers

Palladium toning is much less expensive than gold toning. Ten minutes in 0.05% potassium hexachloropalladate in 4 N hydrochloric acid tones silver-impregnated reticular fibers as well as 3 min in 0.2% aqueous gold chloride does. Differences in toning of the background depend on the silver stain.

Quantification of thalamocortical synapses with spiny stellate neurons in layer IV of mouse somatosensory cortex.

The distribution of thalamocortical (TC) and other synapses involving spiny stellate neurons in layer IV of the barrel region of mouse primary somatosensory cortex (SmI) was examined in seven male CD/1 mice. TC axon terminals were labeled by lesion-induced degeneration, which has been shown to label reliably all TC synapses in mouse barrel cortex. Spiny stellate neurons, labeled by Golgi impregnation and gold toning, were identified with the light microscope prior to thin sectioning and electron microscopy. Analysis of eight dendritic segments from seven spiny stellate neurons showed that most of their synapses are with their dendritic spines, rather than with their shafts. Axospinous synapses are primarily of the asymmetrical type, whereas axodendritic synapses are mainly of the symmetrical type. Dendrites of spiny stellate neurons consistently form thalamocortical synapses, most of which involve spine heads rather than spine stalks or dendritic shafts. From 10.4% to 22.9% of all asymmetrical synapses with dendrites of spiny stellate neurons involve TC axon terminals. In general, this is a higher range than the ranges that characterize the TC synaptic connectivity of dendrites belonging to other types of neurons, implying that spiny stellate neurons are perhaps more strongly influenced by TC synaptic input than other types of cortical neurons examined previously. Spines involved in TC synapses were distributed irregularly along each of the stellate cell dendrites; about half of the interspinous intervals between these spines were about 5 microns or less. Modulations of the efficacy of TC synaptic input to dendrites of layer IV spiny stellate neurons are discussed in the light of recently reported computer simulated analyses of axospinous synaptic connections.