N A N O P R O B E S     E - N E W S

Vol. 9, No. 12          December 31, 2008

The basal ganglia are a group of subcortical nuclei that participate in a number of functions, including motor, cognitive and mnemonic behaviors. Interactions between glutamatergic corticostriatal afferents and dopaminergic nigrostriatal afferents are central to basal ganglia function. The thalamostriatal projection provides a glutamatergic innervation of similar magnitude to the corticostriatal projection, and in their paper in the Journal of Neuroscience, Moss and Bolam use double labeling with silver-enhanced Nanogold and avidin-biotin complex (ABC) peroxidase labeling as part of a study to test the hypotheses that thalamostriatal synapses have similar spatial relationships with dopaminergic axons as corticostriatal synapses do, and that the spatial relationships between excitatory synapses and dopaminergic axons are selective associations.

Rat striatum was immunolabeled to reveal vesicular glutamate transporters (VGluTs) 1 and 2 - markers of corticostriatal and thalamostriatal terminals respectively - together with tyrosine hydroxylase (TH) to reveal dopaminergic axons. 65 µm sagittal brain sections were washed five times in phosphate-buffered saline (PBS) and placed in cryoprotectant (0.05 M phosphate buffer, 25% sucrose, 10% glycerol) for a minimum of 2 hours before freeze-thawing. Each section was immersed in chilled isopentane, then liquid nitrogen for a few seconds, thawed at ~25°C for ~5 minutes, then washed three times in PBS. The sections were then double-immunolabeled to reveal VGluT1 or VGluT2 (markers of cortical and thalamic terminals, respectively) and tyrosine hydroxylase (TH; this is the rate limiting enzyme in the synthesis of catecholamines) as a marker of dopaminergic axons and terminals. In contrast with previous accounts, this time the immunogold-silver staining was completed before peroxidase labeling.

Although TH immunolabeling will also reveal noradrenergic axons, these are rare in the striatum. Normal goat serum in PBS (NGS-PBS; Vector Laboratories) was used to block (10% NGS) and wash (2% NGS) sections before incubation with primary antibodies. Primary antibodies for each of the double immunolabeling experiments were added sequentially. Antibodies against VGluT1 or VGluT2 raised in rabbits were used at a dilution of 1:2000 in 2% NGS-PBS; sections were incubated overnight (15 hours), shaking at room temperature or over three nights (64 h) at 4°C. They were then washed once in 2% NGS-PBS, three times in PBS and once in 1% NGS in PBS. The sections were then incubated for 2 hours in Nanogold goat anti-rabbit antibody at a dilution of 1:100 in 1% NGS-PBS. This was followed by one wash in 1% NGS-PBS, then three washes each in PBS and acetate buffer (0.1 M sodium acetate 3-hydrate) in preparation for silver enhancement. HQ Silver was added to each section and allowed to react for 24 minutes, washed three times each in acetate buffer and PBS, and once in 2% NGS-PBS.

Sections were then incubated in a mouse monoclonal antibody raised against TH (1:1000 dilution of in 2% NGS-PBS) overnight, shaking at room temperature. They were washed once 2% NGS-PBS, three times in PBS and once in 1% NGS-PBS, then incubated with biotin-conjugated goat anti-mouse secondary antibody with shaking at room temperature, for a minimum of 2 hours before one wash in 1% NGS-PBS and three washes in PBS. They were then incubated in avidin-biotin-peroxidase complex (ABC; Vector Laboratories), with shaking for 90 min at ~25°C. After the ABC incubation, sections were washed three times in PBS and twice in Tris buffer (0.05 M, pH 7.4). Diaminobenzidine (DAB; 2.5 mL of 0.025% in Tris buffer) was added to the sections with regular mixing for 15 minutes then 40 µL of H₂O₂ (0.03% in H₂O) was added. The reaction continued for 57 minutes until staining was revealed and the reaction was stopped by the addition of Tris buffer; then, sections were washed twice in Tris buffer and three times in PBS, then into 0.1 M phosphate buffer (PB; pH 7.4).

The primary antibodies were raised against rat VGluT1, VGluT2 and TH (amino acids 543560, 510582 and 40152, respectively). The distribution of immunolabeling at the light and electron microscopic levels was distinct for each primary antibody, and consistent with previous observations. No immunolabeling was observed after omission of the primary antibodies and omission of each of the secondary antibodies individually showed no evidence of cross-reactivity. The sections were then placed flat on the bottom of glass Petri dishes and postfixed in osmium tetroxide (1% in PB; Oxkem) for 7 minutes. They were then washed in 0.1 M PB and dehydrated in an ascending series of ethanol dilutions (15 minutes in 50% ethanol, 35 minutes in 70% ethanol which included 1% uranyl acetate; TAAB; 15 min in 95% ethanol, and twice 15 minutes in absolute ethanol). Then, sections were exposed to two changes of propylene oxide (Sigma) for 15 minutes and lifted, using modified forceps, into resin (Durcupan ACM, Fluka) in crafted foil boats and left overnight (15 hours) at room temperature. The resin was then warmed to decrease its viscosity and sections were placed on microscope slides, a coverslip applied and the resin cured at 65°C for ~70 hours.

For electron microscopic analysis, all sections were first examined in the light microscope and areas from the dorsolateral striatum were cut from the slide, glued to the top of a resin block and trimmed with razor blades. Serial 50 nm sections (silver/gray) were then cut using an ultramicrotome, collected on pioloform-coated, single-slot copper grids, then lead stained to improve contrast for electron microscopic examination.

Over 80% of VGluT-positive synapses were within 1 µm of a TH-positive axon and ~40% were within 1 µm of a TH-positive synapse. Of structures postsynaptic to VGluT1- or VGluT2-positive terminals, 21 and 27%, respectively, were apposed by a TH-positive axon and about half of these made synaptic contact. When structures postsynaptic to VGluT-positive terminals and VGluT-positive terminals themselves were normalized for length of plasma membrane, the probability of them being apposed by, or in synaptic contact with, a TH-positive axon was similar to that of randomly selected structures. Extrapolation of the experimental data to more closely reflect the distribution in 3D reveals that all structures in the striatum are within 1 µm of a TH-positive synapse. Therefore, it was concluded that thalamostriatal synapses are in a position to be influenced by released dopamine to a similar degree to corticostriatal synapses, and these associations arise from a nonselective dopaminergic axon lattice.

References:

• Moss, J., and Bolam J. P.: A dopaminergic axon lattice in the striatum and its relationship with cortical and thalamic terminals. J. Neurosci., 28, 11221-11230 (2008).
  • Abstract (courtesy of the Journal of Neuroscience).

• Bernard, V.; Levey, A. I., and Bloch, B.: Regulation of the subcellular distribution of m4 muscarinic acetylcholine receptors in striatal neurons in vivo by the cholinergic environment: evidence for regulation of cell surface receptors by endogenous and exogenous stimulation. J. Neurosci., 19, 10237-10249 (1999).
  • Abstract (courtesy of the Journal of Neuroscience).
  • Reprint (PDF) (courtesy of the Journal of Neuroscience).

More information:

• Nanogold conjugates: Catalog information
• Nanogold conjugates: Product information, instructions and protocols
• Nanogold conjugates: Technical help
• References: Nanogold labeling, sorted by application
• Silver enhancement: Catalog and general information
• HQ Silver: Product information, instructions and protocols

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Nanogold® Labeling on Surfaces

Although labeling surface features simplifies separation, since the unreacted Nanogold may be removed by washing, the interaction of Nanogold with surfaces can be different to that with biomolecules. In particular, non-specific binding needs to be carefully monitored. All our Nanogold reagents are formulated to be water-soluble, and therefore may mostly be removed by aqueous buffers. However, they do still retain an element of hydrophobic character, and this can result in non-specific interactions with some surfaces. If you find this to be a problem after thorough washing, we recommend the following:

• Organic solvent: on surfaces, the issue of biomolecule denaturation is less likely to be a problem, so you may have more freedom to test organic solvents. First try 50% isopropanol-water, then 100% isopropanol if necessary. If isopropanol does not work, dimethylsulfoxide (DMSO) is an excellent solvent for Nanogold, and should be highly effective in removing it from surfaces. If you are labeling a surface with a biomolecule component, note that you should not use a higher proportion of DMSO than your protein can tolerate: use a DMSO-water mixture with the highest allowable DMSO content. You may need to leave the DMSO in contact with the surface for a few minutes to dissolve any tightly-bound gold.

  Other organic solvents that may be useful are acetonitrile, dimethylformamide (DMF), or N,N-dimethylacetamide (DMA). Mixtures of alcohol (methanol or ethanol) with dichloromethane or with trichloromethane (chloroform) may be useful on surfaces with a more ionic character, such as silica or alumina-based surfaces.

• Triethylammonium bicarbonate is another excellent solvent for Nanogold. It is prepared by bubbling carbon dioxide through a mixture of degassed triethylamine and degassed water; as the carbon dioxide dissolves and the mixture hydrates, the weakly ionic solution is formed. You should prepare a 0.6 M solution in 20% isopropanol; make a more concentrated solution in water first, then dilute in water / isopropanol as appropriate. More details are given in the following reference:

  Safer, D.; Bolinger, L., and Leigh, J. S.: Undecagold clusters for site-specific labeling of biological macromolecules: simplified preparation and model applications. J. Inorg. Biochem., 26, 77-91 (1986).

  • Abstract (courtesy of Science Direct

If the gold is undergoing chemical changes after adhering to the surface (such as forming larger particles) you may need to protect the surface first (using a surfactant or other protective molecule). Formation of larger gold particles is evidenced by a color change of the surface to red or purple, or the formation of a gray or black precipitate.

More information:

• Technical help: Nanogold labeling reagents
• Guide to Gold Cluster Labeling
• Catalog information: Nanogold labeling reagents
• Product instructions: Monomaleimido Nanogold
• Product instructions: Mono-Sulfo-NHS-Nanogold
• Product instructions: Monoamino Nanogold
• References sorted by application - Nanogold labeling

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AuroVist: More Contrast for X-Ray Imaging

AuroVist is a stabilized 1.9 nm gold particle. It provides better contrast than iodine for both micro-CT and clinical CT applications.* At appropriate beam energies, gold achieves a contrast up to three times greater than iodine per unit mass, yielding initial blood contrast greater than 500 Hounsfeld Units (HU). Gold concentrations up to four times those of iodine can be achieved, providing a total contrast gain of up to ten times or more. In addition, AuroVist gives you these enhanced performance features:

* Research use only. Not approved for clinical or human use.

We have received several inquiries about whether a targeted version of this reagent is available. The preparation of a targeted gold nanoparticle reagent on a sufficient scale for X-ray contrast imaging is challenging both because of the greater complexity of the synthesis, and also because the amount of reagent required for visualization depends on the size and target density of the feature to be imaged, and how effectively the targeting mechanism can deliver a visible dose to the target. However, we are working to develop this technology, and hope to incorporate it into future AuroVist products.

AuroVist is a new product, and therefore it has not been optimized in all possible applications. However, the following guidelines may be helpful in obtaining the best results with this reagent.

Best instrument and beam settings

The absorption increases by a significant factor (jump ratio) above the gold L and K edges. The X-ray properties of gold, showing the jump ratios for these regions, are shown in the table below. It is therefore advantageous to image using these absorptions. The settings below are appropriate for the different instruments.

This must be compared this with the absorption spectrum of soft tissue. X-ray absorption spectra for elements and tissues are available from NIST:

X-ray absorption spectra

How to ensure minimal toxicity

Certain strains of mice appear to be more tolerant of this gold. For Balb/C, the LD50 is ~ 3.2 g Au/kg. Nude mice and C3H mice also seem to respond about the same. Some outbred mice, however, appear to have a lower LD50 of about > 1.4 g Au/kg. If you are using outbred mice, or a different strain to those mentioned above, it is recommended that you use this lower value. The following suggestions may be helpful:

• Start with a moderate dose, such as 120 or 160 mg/mL. The value of 270 mg/mL reported in our paper in the British Journal of Radiology was the highest value tested; at high concentrations acute toxicities increase rapidly, and a modest reduction in dose can significantly reduce toxicity without compromising your results.

• Centrifuge and then filter the reconstituted reagent through a sterile filter immediately before injection to ensure that no aggregates or larger particles are present. Larger particles and aggregates can impact biocompatibility, and may also have reduced stability and hence a higher tendency to deposit in tissues and organs, with negative consequences.

• Use one of the pure-bred mouse strains mentioned above, which are known to have higher tolerance. If you are planning to conduct multiple experiments using a different strain, it may be worthwhile to test your strain first.

References:

• Hainfeld, J. F.; Slatkin, D. N.; Focella, T. M, and Smilowitz, H. M.: Gold nanoparticles: a new X-ray contrast agent. Br. J. Radiol., 79, 248-253 (2006).
  • Abstract (courtesy of the British Journal of Radiology).

• Hainfeld, J. F.; Slatkin, D. N.; Focella, T. M., and Smilowitz, H. M.: In Vivo Vascular Casting. Microsc. Microanal., 11, (Suppl. 2: Proceedings); Price, R.; Kotula, P.; Marko, M.; Scott, J. H.; Vander Voort, G. F.; Nanilova, E.; Mah Lee Ng, M.; Smith, K.; Griffin, P.; Smith, P., and McKernan, S., Eds.; Cambridge University Press, New York, NY, p. 1216CD (2005).
  • Paper

• Hainfeld, J. F., Slatkin, D. N., and Smilowitz, H. M.: The use of gold nanoparticles to enhance radiotherapy in mice. Phys. Med. Biol., 49, N309-N315 (2004).
  • Abstract (courtesy of Physics in Medicine and Biology).

More information:

• AuroVist: Catalog information
• AuroVist: Product information, instructions and protocols
• Technical help: AuroVist
• AuroVist: References sorted by application
• In Vivo Vascular Casting: our paper from Microscopy and Microanlaysis 2005

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Nanogold® and GoldEnhance Track Cell Migration

Gold enhancement has important advantages over silver enhancement for several applications:

• Cleaner signals with lower background: for light microscopy and blotting, GoldEnhance offers both high sensitivity and improved signal clarity, with lower background, than silver enhancement.

• Osmium etch resistance for EM: gold enhancement may safely be used before any strength osmium tetroxide - gold enhanced particles are not etched as silver can be.

• Physiological compatibility: Gold enhancement may be used in physiological buffers such as phosphate-buffered saline without the risk of precipitation (halides, such as chloride, precipitate silver).

• Metal substrate compatibility: gold enhancement may be conducted in the presence of metals (such as metallic substrates for cell culture or biomaterials).

• pH compatibility: the metallographic reaction is less pH sensitive than that of silver.

• Better SEM Visualization: gold gives a much stronger backscatter signal than silver, making it better for SEM labeling applications.

• Mild and easy to use: GoldEnhance is near neutral pH for best ultrastructural preservation, and has low viscosity so the components may be dispensed and mixed easily and accurately.

Gold enhancement has been used for in situ hybridization, double labeling by sequential enhancement of Nanogold®, and for electron microscopic visualization and tracking of cellular uptake of gold nanoparticles for drug delivery; it also provides cleaner, more sensitive detection than silver enhancement on western blots and dot blots. In their recent paper in the Journal of Cell Biology, Kriebel and co-workers show that it remains a highly effective tool for electron microscopy, using it show the cellular distribution of adenylyl cyclase (ACA) and its role in cell mobility.

Chemoattractant signaling induces the polarization and directed movement of cells, secondary to the activation of multiple effector pathways. Chemotactic signals can also be amplified and relayed to proximal cells via the synthesis and secretion of additional chemoattractant. The mechanisms of these processes remain ill-defined. The ability of Dictyostelium cells to spontaneously aggregate and stream relies on the presence of a finely regulated signal relay loop that is centered on cAMP. In this organism, the detection, synthesis, and degradation of cAMP are highly regulated. Addition of chemoattractants leads to a burst in the activity of adenylyl cyclase (ACA).

Immunogold EM staining was used to localize a YFP-ACA fusion protein, thus enabling combined fluorescent and immunogold labeling of the target sites. Cells were differentiated and prepared for microscopy, and fixed at room temperature for 15 minutes in 1% formaldehyde, 0.1% glutaraldehyde, and 0.01% digitonin in 15 mM Pipes/1 mM EGTA, followed by 15 minutes in 1% formaldehyde. After blocking in 50 mM NH₄Cl, 0.1% digitonin, and 1% BSA in PBS, the cells were incubated with an anti-GFP antibody (1:500; Abcam) in block solution overnight at 4°C followed by Nanogold anti-rabbit Fab fragments. Staining was enhanced for 7 minutes with GoldEnhance (Nanoprobes) made with one part component A, one part component B, one part component C, and three parts PBS. Afterward, the samples were stained with osmium and embedded in epon. Images were taken with a transmission microscope.

An asymmetrical distribution of adenylyl cyclase (ACA) was found at the back of Dictyostelium discoideum cells. This is an essential determinant of their ability to migrate in a head-to-tail fashion, and requires vesicular trafficking. This trafficking results in a local accumulation of ACA-containing intracellular vesicles and involves intact actin, microtubule networks, and de novo protein synthesis. The results also show that migrating cells leave behind ACA-containing vesicles, likely secreted as multivesicular bodies and presumably involved in the formation of head-to-tail arrays of migrating cells. The authors propose that similar compartmentalization and shedding mechanisms exist in mammalian cells during embryogenesis, wound healing, neuron growth, and metastasis.

Reference:

• Kriebel, P. W.; Barr, V. A.; Rericha, E. C.; Zhang, G., and Parent, C. A.: Collective cell migration requires vesicular trafficking for chemoattractant delivery at the trailing edge. J. Cell Biol., 183, 949-961 (2008).
  • Abstract (courtesy of Journal of Cell Biology).

More information:

• Gold Enhancement: introduction and catalog information
• GoldEnhance EM: instructions and product information
• GoldEnhance LM/Blot: instructions and product information
• Gold-Based Autometallography: our paper from Microscopy & Microanalysis 1999
• Tech help: gold enhancement
• References: gold enhancement

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Nanoprobes: New Year Hours - and Order Now at 2008 Prices

Unfortunately, our costs have increased over the last few months, and as a result, we will need to raise our prices slightly. Therefore, if you plan to order our products, you can avoid the increase by placing your orders immediately.

More information:

• Contact information
• Nanoprobes: news and press releases
• About Us: company information
• Our story: company history

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Other Recent Publications

References:

• Zheng, J.; Zhang, C., and Dickson, R. M.: Highly fluorescent, water-soluble, size-tunable gold quantum dots. Phys. Rev. Lett., 93, 077402 (2004).
  • Abstract (courtesy of Physical Review Letters). ).
• Zheng, J.; Nicovich, P. R., and Dickson, R. M.: Highly fluorescent noble-metal quantum dots. Annu. Rev. Phys. Chem., 58, 409-431 (2007).
  • Abstract (courtesy of Annual Reviews of Physical Chemistry). ).

Reference:

• Reil, F.; Hohenester, U.; Krenn, J. R., and Leitner, A.: Forster-Type Resonant Energy Transfer Influenced by Metal Nanoparticles. Nano Letters, 8, 4128-4133 (2008).
  • Abstract (courtesy of ACS Publications).

In the same issue of Nano Letters, Bigall and co-workers presented a facile, reproducible method for synthesizing monodisperse platinum (Pt) nanoparticles with sizes ranging from 10 to 100 nm in diameter. These particles were highly monodisperse, with standard deviations of 3% for the larger sizes. The reaction takes place in aqueous solution using a multistep, seed-mediated approach. Seeds were prepared by reduction of chloroplatinic acid with sodium citrate solution, followed quickly by sodium borohydride. Larger particles were prepared by addition of these seeds to solutions of hexachloroplatinic acid in sodium citrate / ascorbic acid solution. The Pt nanospheres were found to consist of several small crystallites resulting in a surface roughness of 5-10 nm. Extinction spectra are measured from particles dispersed in water and calculated for single particles which are found to be in excellent agreement. A linear correlation was obtained between the plasmon extinction maximum (from UV to the visible regions) and the particle diameter, providing a potentially useful tool for identification and monitoring of such nanoparticles.

Reference:

• Bigall, N. C.; Härtling, T.; Klose, M.; Simon, P.; Eng, L. M., and Eychmüller, A.: Monodisperse Platinum Nanospheres with Adjustable Diameters from 10 to 100 nm: Synthesis and Distinct Optical Properties. Nano Letters, 8, 4588-4592 (2008).
  • Abstract (courtesy of ACS Publications).

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