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N A N O P R O B E S     E - N E W S

Vol. 7, No. 12          December 21, 2006


Updated: December 21, 2006

In this Issue:

This monthly newsletter is to inform you about techniques to improve your immunogold labeling, highlight interesting articles and novel applications of metal nanoparticles, and answer your questions. We hope you enjoy it and find it useful; as always, let us know if we can improve anything.

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Nanogold® in Neuroscience: Spinal Cord Interneurons and Potassium Channels

If you are a neuroscience researcher, you may be aware of the extensive use of Nanogold® to identify and localize neuronal features, as well as its combination with enzymatic labeling to provide an immunoelectron microscopic double labeling method. Cheryl Marker, Rafael Lujan and colleagues took the field a little further recently with their use of Nanogold to study the distribution of G-protein gated potassium channels among spinal cord neurons and determine the neuronal circuitry of the spinal cord, reported recently in the Journal of Neuroscience.

Noxious stimuli are sensed and carried to the spinal cord dorsal horn by Adelta and C primary afferent fibers. Some of this input is relayed directly to supraspinal sites by projection neurons, while much of it impinges on a heterogeneous population of interneurons in lamina II. G-protein-gated inwardly rectifying potassium (GIRK) channels have been shown to be expressed in lamina II of the mouse spinal cord, and pharmacologic ablation of spinal GIRK channels has been found to selectively blunt the analgesic effect of high - but not lower - doses of intrathecal µ-opioid receptor (MOR) agonists.

GIRK channels formed by GIRK1 and GIRK2 subunits have now been found in two large populations of lamina II excitatory interneurons. Electrophysiological studies showed that one population displays relatively large apparent whole-cell capacitances and prominent GIRK-dependent current responses to the MOR agonist [D-Ala2,N-MePhe4,Gly-ol 5]-enkephalin DAMGO). A second population shows smaller apparent capacitance values, and exhibits a GIRK-dependent response to the GABAB receptor agonist baclofen, but not to DAMGO.

Immunoelectron microscopy with Nanogold® labeling was used to localize the GIRK subunits and determine their distribution in these two populations, via ultrastructural analysis using a pre-embedding approach. Free-floating sections were incubated in 10% normal goat serum (NGS) diluted in Tris-buffered saline (TBS) for 1 hour at room temperature, then incubated for 48 hours at 4°C with primary antibodies (GIRK1 or GIRK2) at final concentrations of 12 µg/ml diluted in TBS/1% NGS. GIRK subunit immunoreactivity was revealed by silver-intensified immunogold reaction: sections were incubated for 3 hours at room temperature with goat Nanogold-Fab' anti-rabbit or biotinylated goat anti-rabbit Fab fragments. All secondary antibodies were diluted 1:100 in TBS/1% NGS. Sections were then washed with TBS and double-distilled water, followed by silver enhancement of the gold particles with HQ Silver for 810 min. After washing in PBS and postfixing with OsO4 (1% in 0.1 M phosphate buffer) and block staining with uranyl acetate, the sections were dehydrated in graded series of ethanol solutions, and flat-embedded on glass slides in Durcupan resin. Regions of interest were cut at 7090 nm with an ultramicrotome. Ultrathin sections were mounted on 200-mesh nickel grids, and counterstained on drops of 1% aqueous uranyl acetate, followed by Reynoldss lead citrate. Electron microscopic samples were obtained from three different mouse spinal cords, and three blocks of each animal were cut for electron microscopy.

Ultrastructural analysis was performed with a TEM-JEOL 100-CX electron microscope (JEOL, Peabody, MA), and photomicrographs captured with a CCD camera and digitally modified for brightness and contrast. To assess the specificity of GIRK subunit antisera, corresponding sections from GIRK knock-out mice were processed in parallel. Labeled structures were classified as axons, axon terminals, synapses, dendrites, or astrocytic processes based on morphological information in each section: axons were identified by their lack of synaptic input, the presence of neurotubules and occasional vesicles, and, in rare instances, presence of myelin. Axon terminals were identified by the presence of synapses and small round and/or large granular vesicles and synapses identified as parallel membranes separated by widened clefts associated with membrane specializations. Synapses displaying a prominent density on the postsynaptic side were characterized as asymmetrical (putative excitatory), while those showing equivalent densities on both sides were characterized as symmetrical (putative inhibitory). Smaller axon terminals that contained flattened vesicles and establish a single synapse were also identified in lamina II. These structures are thought to represent the terminal endings of intrinsic GABAergic interneurons. Dendrites were identified by the presence of synaptic contacts, lack of small vesicles, diffuse filaments, and numerous mitochondria. Astrocytic processes were identified by their amorphous shape, lack of vesicles and synaptic contacts, and presence of glial microfilaments.

Ultrastructural analysis revealed that GIRK subunits preferentially label type I synaptic glomeruli. This suggests that GIRK-containing lamina II interneurons receive prominent input from C fibers, and little input from Adelta fibers. Thus, excitatory interneurons in lamina II of the mouse spinal cord can be subdivided into different populations based on the neurotransmitter system coupled to GIRK channels. This important distinction provides a unique opportunity to characterize and relate spinal nociceptive circuitry according to a defined physiological significance.

Reference:

  • Marker, C. L.; Lujan, R.; Colon, J., and Wickman, K.: Distinct populations of spinal cord lamina II interneurons expressing G-protein-gated potassium channels. J. Neurosci., 26, 12251-12259 (2006).

More information:

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Troubleshooting: Low or No Labeling with Nanogold® Conjugates

I just conducted an immunogold staining experiment with a Nanogold®-Fab' or IgG conjugate, but when I examined the specimen, I could not see any gold particles or any evidence of staining. What should I do?

There are several reasons for low labeling, or an apparent failure to label, and the failure may be due to one of several components. Working through the following checklist will help you to identify the cause of your low labeling in the most efficient manner, losing the least amount of time.

  1. Does the labeling work with other probes? For example, have you successfully labeled the same target using the same primary antibody and a fluorescent or enzymatic probe? If so, then you know that the target or the primary antibody to which your Nanogold® conjugate is binding is intact and working as it should. In this case, you should check the Nanogold conjugate and labeling procedure, and any reagents applied afterwards, such as silver enhancers, gold enhancers, or poststains.

  2. If you are using silver or gold enhancement, are these reagents working? If you can test them in an experiment where you have used another immunogold probe that is known to be good, this will rule these out as the source of your low labeling.

  3. Does the Nanogold reagent appear as it should? Nanogold conjugates in solution are a clear, pale brown to greenish-brown. If the color has changed, particularly if it is red, purple, blue, or gray, this is an indication that the Nanogold may be degraded. To be sure, check the conjugate by UV/visible spectroscopy. The UV/visible absorption spectrum of a Nanogold conjugate is shown below. Because antibodies do not absorb at wavelengths higher than about 300 nm, the region from 320 nm to 800 nm, marked in red as the Nanogold absorbance profile, will be the same for all Nanogold conjugates (note that because bovine serum albumin is added to stabilize the solution, absorbance at 280 nm will be higher for commercial Nanogold conjugates than is shown).

    [Nanogold-Fab' conjugate: UV/visible absorption spectrum (7k)]

    UV/visible absorption spectrum of a Nanogold-Fab' conjugate. The region marked in red, the, "Nanogold absorbance profile," is the region in which only Nanogold absorbs: neither the antibody nor the additional bovine serum albumin added to stabilize the conjugate absorbs in this region. Therefore this profile should be similar in all Nanogold conjugates.

    If you observe a profile that is significantly different - especially the appearance of a broad maximum between 500 and 550 nm, or a significant increase in absorbance at longer wavelengths - this indicates decomposition of the Nanogold particle. Additional spectra are available in our Guide to Gold Cluster Labeling on our web site.

    If the spectrum appears to be normal, then you can check the integrity and reactivity of the Nanogold conjugate using blot tests, as described below.

  4. Checking conjugate integrity: is the Nanogold still attached? You can check whether the Nanogold particle is still attached to the antibody using a simple blot test. Spot 1 µL of the Nanogold conjugate onto a nitrocellulose membrane, air-dry for 15 minutes, then develop with LI Silver (30 minutes), HQ Silver (20 minutes), GoldEnhance EM (5 minutes), or a silver enhancement reagent from another manufacturer (develop as directed for dot blots). If the Nanogold label is intact and still conjugated, you will see a brown to black spot develop. If you do not see this and you know that the silver or gold enhancement reagent is good, the Nanogold has dissociated; please contact us.

    If you obtain a strong signal in this blot, the problem may lie with the antibody part of the Nanogold conjugate. You can test this as described below.

  5. Is the Nanogold conjugate binding to its target? To check whether the immunoreactivity of the Nanogold conjugate has been compromised, blot 1 µL of your primary antibody (0.1 - 1 mg/mL is a good concentration to use) onto a nitrocellulose membrane. Block with 4% bovine serum albumin (BSA) in phosphate-buffered saline (PBS), pH 7.4, for 30 minutes at 37°C, then wash with 1% BSA in PBS for 5 minutes. Incubate for one hour with a 1 : 50 dilution of the Nanogold conjugate, also in 1% BSA in PBS. Wash three times with 1% BSA in PBS, then three times with PBS, then twice with deionized water. Develop with silver or gold enhancement as described above. A brown or black spot confirms reactivity of the Nanogold conjugate towards your primary.

    If you obtain strong reactivity with this test, then you will need to revise your procedure. Try higher concentrations or primary antibody and Nanogold conjugate, longer incubation (overnight), or a higher degree of permeabilization before application; once you have achieved successful labeling, you can then gradually reduce the degree of permeabilization if you require better ultrastructural preservation.

    It should be noted that different antibodies, or antibodies with the same specificity from different suppliers, can differ in their reactivity, and we have occasionally found that antibodies we use for conjugation may be only weakly reactive towards some primaries. If this is the case, you may need to modify your labeling scheme to include a more widely used secondary, and use the Nanogold conjugate as a tertiary labeled probe.

How stable is Nanogold? How can I check whether my conjugates are still good?

Because they are covalently linked rather than adsorbed, Nanogold conjugates are highly stable. Provided they are not allowed to dry out or become contaminated, they can last well beyond the nominal year's shelf life if they are refrigerated at 4°C; we have tested them after two or three years and found blot detection sensitivity to be unchanged (Nanogold conjugates include low levels of a bacterial inhibitor).

Dot blots, as described above, are an excellent test for reactivity of Nanogold conjugates. For product testing purposes, we consider clear detection of 10 ng of target after silver enhancement to be acceptable (In practice, we can usually detect 0.1, and frequently 0.01 ng of target after silver enhancement), so a dot blot which develops a clear signal with 1 or 5 ng of target (or primary antibody) is a good indication of strong binding. However, the spots do become progressively lighter with smaller amounts of target, and if you compare your blots with one that shows how the spots look for a series of dilutions of target, it will give you a good qualitative idea of how good your conjugates are.

Two examples of different dot blots with Nanogold conjugates are shown below to illustrate the range of spot intensities you might encounter. In (b) the first (darkest) pair of spots (top left) contain 10 ng of target IgG; reading left to right along the top row, the second pair contains 5 ng, and the third 1 ng. If your test spots with 1 or 5 ng of target look like any of these, your conjugate is working well. After silver or gold enhancement, the spots should be black (sometimes purplish, sometimes brownish), rather than the deep red found with colloidal gold.

Our experience with conventional colloidal gold is more limited, but for conjugates of 5, 10 and 15 nm colloidal gold, the development of a clear red spot with 50 ng of target is generally a good indication that the conjugate is intact and working as it should. As shown above, you should see progressively lighter spots down to about 5 or 10 ng, sometimes 1 ng for 15 nm gold, for good preparations. For larger gold preparations, the spots will be darker. A deep cherry-red spot is a good indication of a working conjugate.

[Nanogold and colloidal gold blots illustrating detection sensitivity (63k)]

Blot test with Nanogold and colloidal gold conjugates. (a) dot blot image of Nanogold-Fab' Goat anti-Rabbit IgG, developed with GoldEnhance EM gold enhancement reagent, using the Schleicher & Schuell (recently acquired by Whatman) MINIFOLD-1 Dot Blot System which applies the target precisely over a circle with a diameter of 5 mm. The 5 mm circle is big enough for a quantitative analysis by a densitometer. With the target spreading over 5mm circle, we can detect as low as 0.01 ng antigen. While we use a pipette to apply 1 L of target over a circle of 1-2 mm, we can detect as little as 0.005 ng by the same NG conjugates. (b) Nanogold anti-mouse Fab' blotted against mouse IgG, developed with LI Silver (Nanoprobes), showing sensitivity enhancement with smaller spot size. Target was applied using a 1 µL microcapillary tube. This immunodot blot shows 0.1 pg sensitivity (arrow). (c) Colloidal gold blot using a 15 nm colloidal gold conjugate to detect serial dilutions of an IgG target, without silver or gold enhancement. The range of color densities should give a good basis for comparing your results - i.e. if your spot is darker than our 50 ng spot your conjugate is working very well, but if it is much fainter, it is questionable.

More images are available on our web site and in Hainfeld and Furuya's paper describing Nanogold, below.

For extended storage, we do not usually freeze Nanogold conjugates. If you need to do so, freezing at -20°C in 30% glycerol is recommended in order to avoid ice crystal damage.

Reference:

  • Hainfeld, J. F., and Furuya, F. R.: A 1.4nm Gold cluster covalently attached to antibodies improves immunolabeling. J. Histochem. Cytochem., 40, 177-184 (1992).

More information:

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FluoroNanogold: Check your Labeling before EM Processing

If you want to check labeling in your system before you process specimens for electron microscopy, you can - with FluoroNanogold, our unique combined fluorescent and gold labeled immunoprobe. This probe contains both the 1.4 nm Nanogold® label and a fluorescent label (currently Alexa Fluor®* 488 or 594, or fluorescein, are available; other fluorescent labels are planned), both covalently linked to Fab' fragments to give a probe with the same high penetration and antigen access of Nanogold-Fab' fragments. Our Alexa Fluor®* FluoroNanogold probes offer superior fluorescence labeling performance:

  • Increased fluorescence brightness and higher quantum yield.
  • Improved solubility: lower background signal and higher signal-to-noise ratios.
  • Fluorescence remains high and consistent across a wider pH range.

Since we now offer both Alexa Fluor®* 488 and Alexa Fluor®* 594 FluoroNanogold, you can now use these probes to differentiate multiple targets using different colored fluorescence.

In addition to its use for combined and correlative fluorescence and transmission electron microscopic labeling, the combined fluorescent and gold probe FluoroNanogold is useful for a number of other correlative microscopy methods. In recent articles, we have highlighted its use for correlative fluorescence and scanning electron microscopy, and for correlative optical and X-ray fluorescence microscopy. The structure of Alexa Fluor®* 488 FluoroNanogold-Fab', and some results obtained with it, are shown below.

[Alexa Fluor 488 FluoroNanogold-Fab' and results with it (87k)]

Left: Structure of Alexa Fluor®* 488 FluoroNanogold - Fab' and Streptavidin, showing covalent attachment of components. Center: Fluorescent staining obtained using Alexa Fluor 488 FluoroNanogold as a tertiary probe to label red blood cells. Specimen is a slide from the NOVA Lite ANA HEp-2 test, an indirect immunofluorescent test system for screening anti-nuclear antibodies in human serum, stained using positive pattern control human sera, a Mouse anti-Human secondary antibody, and Alexa Fluor 488 FluoroNanogold tertiary probe. Specimens were washed (PBS, 30 minutes) between each step, then blocked by addition of 7% nonfat dried milk to the tertiary antibody solution (original magnification x 400). Right: Scanning electron micrograph of a peg-like terminal constriction of an Oziroë biflora (plant, Hyacinthaceae) chromosome. The image shows both chromosome topography (secondary electron signal) and hybridized enhanced gold signals (superimposed back-scattered electron signals, yellow) labeling 45S rDNA in the nucleolus organizing region with Alexa Fluor®* 488 FluoroNanogold-Streptavidin (micrograph courtesy of Elizabeth Schröder-Reiter and Gerhard Wanner)

Aggregated, highly phosphorylated tau protein is a pathological marker of Alzheimers disease (AD) and other tauopathies. Iliev and co-workers recently used FluoroNanogold as part of their ultrastructural investigations into the effects of modifications to the amino acid sequence motifs in the microtubule-binding repeats of tau. In native tau, motifs of alternating polar and apolar amino acids within the microtubule-binding repeats are interrupted by small breaking stretches. Minimal mutation of these breaking sequences yielded a unique tau mutant containing longer stretches of polar/apolar amino acids which aggregated instantly without losing its microtubule-binding capacity. Immunofluorescence and fluorescence recovery after photobleaching (FRAP) studies showed that these modifications produced rapid aggregation and cytotoxicity with accompanying occurrence of pathologic tau phosphoepitopes (AT8, AT180, AT270, AT100, Ser422, and PHF-1) and conformational epitopes (MC-1 and Alz50) in CHO cells.

Aggregation was confirmed at the ultrastructural level using FluoroNanogold for pre-embedding electron microscopic analysis of Sarkosyl extracts and CHO cells. Sarkosyl extract samples were absorbed on Formvar carbon-coated grids and counterstained with 2% (w/v) aqueous uranyl acetate. For electron microscopy on cells, the cells were fixed 24 h after transfection using 2% (w/v) formaldehyde and 0.25% (v/v) glutaraldehyde (EM grade) in PBS at pH 7.4, for 30 minutes at room temperature. Pre-embedding labeling was conducted to identify the transfected cells: cells were permeabilized with 0.1% Triton X-100, then blocked with 1% bovine serum albumin, fraction V, with 0.045% (w/v) cold water fish gelatin in PBS. Pre-embedding immunogold labeling was conducted using the anti-C-terminal tau antibody (clone T46: 1:400) as the primary, and Nanogold - and Alexa Fluor 488 - conjugated goat Fab' anti-mouse IgG, diluted 1:30 in PBS with 0.05% Triton X-100 for 1 hour. Silver enhancement of the Nanogold particles was performed using LI Silver enhancement reagent. Cells were then gently scraped in PBS, collected, and postfixed with 1% (w/v) osmium tetroxide. The fixed cells were dehydrated in a graded series of ethanol containing 0.5% uranyl acetate, then embedded in Araldite by passage through propylene oxide and propylene oxide-Araldit (1:1) solutions followed by polymerization at 60°C. Ultrathin sections were cut, mounted on Formvar-coated copper grids, and counterstained with 0.4% lead citrate. All samples were analyzed on a Zeiss EM 10B electron microscope at an accelerating voltage of 60 kV.

3PO-tau phosphorylation was also assessed in human SH-SY5Y neuroblastoma cells, which natively express several tau isoforms and represent an experimental system closer to neurons. A significant increase in the phosphorylation level of 3PO-tau was found in both CHO fibroblasts and SH-SY5Y human neuroblastoma cells, at sites that correspond to known pathological hyperphosphorylation epitopes. Similar to pathological tau in the pretangle state, toxicity appeared to occur early without the requirement for extensive fibril formation. FRAP analysis demonstrated that 3PO-tau can recruit wild type (WT)-tau protein into its aggregates: WT-tau showed a significantly decreased motility in 22% of the cells. Co-aggregation was either almost complete, or absent: most cells co-expressing 3PO-tau did not affect the diffusion properties of WT-tau-YFP (and YFP alone), even in cases where WT-tau seemed to co-localize with the aggregated 3PO-tau. This shows that 3PO-tau aggregation does not greatly affect the inherent diffusion of cytosolic proteins or act as a steric nonspecific protein trap, and show that this approach is useful for the identification of proteins that are specifically recruited to aggregated tau to mediate pathological cellular processes. This mutant protein therefore provides a novel platform for the investigation of the molecular mechanisms for toxicity and cellular behavior of pathologically aggregated tau proteins and the identification of its interaction partners.

Reference:

  • Iliev, A. I.; Ganesan, S.; Bunt, G., and Wouters, F. S.: Removal of pattern-breaking sequences in microtubule binding repeats produces instantaneous tau aggregation and toxicity. J. Biol. Chem., 281, 37195-37204 (2006).

More information:

  • Pre-embedding Nanogold immunolabeling with HQ Silver enhancement: special feature

    * Alexa Fluor is a registered trademark of Invitrogen (Molecular Probes), Inc.

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    GoldEnhance: Better Enhancement for Nanogold® and Colloidal Gold

    Gold enhancement is an autometallographic method, similar to silver enhancement, in which gold is deposited onto gold nanoparticles. It has significant advantages for both scanning electron microscopy (SEM) and transmission electron microscopy (TEM):

    • Gold enhancement may safely be used before any strength osmium tetroxide - it is not etched.
    • May be used in physiological buffers (including chlorides, which precipitate silver, but not gold).
    • The metallographic reaction is less pH sensitive than that of silver.
    • Gold gives a much stronger backscatter signal than silver.
    • GoldEnhance is near neutral pH for best ultrastructural preservation.
    • Low viscosity, so the components may be dispensed and mixed easily and accurately.
    Kim and group recently demonstrated the advantages of gold enhancement for SEM, and showed that gold enhancement with our GoldEnhance reagents not only works well with Nanogold®, but also with 10 nm colloidal gold to give uniform, dense and readily visualized particles. In their recent paper in Plant Physiology on the existence and role of extracellular ATP (eATP) in plants, they used a novel reporter with gold-enhanced immunogold to identify eATP and localize its release to domains associated with root hair growth and calcium gradients.

    Extracellular ATP (eATP) in animals plays a well-known and documented role in cellular signaling, but although the existence of eATP has been postulated in plants, up until now there has been no definitive experimental evidence for its presence, or an explanation as to how such a polar molecule could exit the plant cell and what physiological role it might play in growth and development. The presence of eATP in plants (Medicago truncatula) was detected by constructing a novel reporter which consisted of a cellulose-binding domain (CBD) peptide to the ATP-requiring enzyme luciferase. When this reporter was applied to plant roots, it enables visualization of eATP in the presence of the substrate luciferin. Luciferase activity was detected in the interstitial spaces between plant epidermal cells, predominantly at the regions of actively growing cells: elevated eATP levels were closely correlated with regions of active growth and cell expansion.

    The CBD and luciferase domain were localized using immunofluorescence and Immunogold Detection. Immunofluorescence detection was conducted in whole-mount preparations; root segments were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde, washed, extracted in 100% ice-cold (-20°C) methanol, rehydrated, blocked with goat serum, then incubated in either 1:200 monoclonal anti-CBD antibody (mouse, clone CBD-8) or monoclonal luciferase antibody (mouse, clone LUC-1), diluted in blocking buffer (2% IgG-free goat serum, 2% IgG-free bovine serum albumin, and 0.1% Micro-O-Protect), and incubated overnight. After washing, samples were incubated in an anti-mouse secondary antibody conjugated with Alexa Fluor® 568, diluted 1:500 in blocking buffer, mounted in an antifade mounting medium (Mowiol) and imaged under the confocal microscope using a 568-nm excitation line (600/40 emission) of a Kr/Ar mixed-gas laser. For immunoelectron microscopy, samples labeled with monoclonal antiluciferase antibody were processed with another secondary anti-mouse antibody conjugated with 10 nm gold, then imaged either directly or after gold enhancement, either in the same confocal microscope using a polarization filter in the path of a 488-nm Kr/Ar laser line (reflection mode), or under a scanning electron microscope.

    The role of eATP was probed using pharmacological compounds known to alter cytoplasmic calcium levels: this revealed that ATP release is a calcium-dependent process, and may occur through vesicular fusion, an important step in the polar growth of actively growing root hairs. Reactive oxygen species (ROS) activity at the root hair tip is essential for root hair growth, and also dependent on cytoplasmic calcium levels. Application of exogenousATP and a chitin mixture increased ROS activity in root hairs, no changes were observed in response to adenosine, AMP, ADP, and nonhydrolyzable ATP (bgmeATP); however, application of exogenous potato (Solanum tuberosum) apyrase (ATPase) decreased ROS activity. This suggests that cytoplasmic calcium gradients and ROS activity are closely associated with eATP release.

    Reference:

    • Kim, S. Y.; Sivaguru, M., and Stacey, G.: Extracellular ATP in plants. Visualization, localization, and analysis of physiological significance in growth and signaling. Plant Physiol., 142, 984-992 (2006).

    In some situations, GoldEnhance may produce a fine, "granular" non-specific background signal of small particles. There are several ways in which you can either slow down development so that you have more control over its progress, or chemically "stop" the development process. Which approach is best for the system under study depends upon whether you believe the primary problem is continued development after rinsing, in which case a chemical "stop" is appropriate, or excessively fast development before rinsing, in which case slowing down the reaction is most appropriate.

    If you are observing fine background that seems to arise from continued development after rinsing to remove the GoldEnhance reagents, a number of "stop" procedures are available. The simplest and most universal is treatment with 1% or 2% freshly prepared sodium thiosulfate for a few seconds, and unless there are specific reasons that this would be harmful to your specimen, we usually suggest that you try this first.

    Wanzhong He, working in the laboratory of Dr. Pamela J. Bjorkman in the Division of Biology at the California Institute of Technology has been kind enough to send us some results using a combination of methods that eliminated background to produce very clean staining:

    • A ratio of one part solution A (enhancer) : 5 parts solution B (activator) was used.
    • Solution D (buffer) was substituted with 0.05 M sodium phosphate with 0.1 M sodium chloride, pH 5.5.
    • Enhancement was performed for 10-15 minutes rather than 3 minutes.
    • The reaction was "stopped" using 3% Na2S2O3 for 30 seconds, then washed with 10% acetic acid + 10% glucose for 3 x 5 minutes.

    Some preliminary images are shown below:

    [Effect of modifications to control background staining with gold enhancement (155k)]

    left: Gold enhancement of intestinal tissue with Nanogold staining. (a) Nanogold particles, then enhanced with GoldEnhance EM for 3 minutes; (b) control with Nanogold labeling omitted, showing the granular background. right: analogous procedure, using modified protocol to control background, comprising: one part A (enhancer) : 5 parts B (activator); D (buffer) was substituted with 0.05M sodium phosphate with 0.1M sodium chloride, at pH 5.5; enhancement was performed for 10-15 minutes rather than 3 minutes, "stopped" using 3% Na2S2O3 30 seconds, then washed with 10% acetic acid + 10% glucose for 3 x 5 minutes. (c) Nanogold particles, enhanced with GoldEnhance EM; (d) control with Nanogold labeling omitted, identical gold enhancement procedure. Insets show experimental (left) and control (right) specimens (Thanks to Wanzhong He and Dr. Pamela Bjorkman for the images).
    We have extensively discussed alternative strategies for controlling background in a previous article and on our web site.

    More information:

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    Nanoprobes Holiday Hours

    Nanoprobes will be closed on Monday, December 25, and Monday, January 1. We will be open for business on all other days, and you can place orders in the usual way. Orders placed on these days will be shipped the following business day provided the items are in stock. We will have fewer staff to answer the telephone, so please leave a message if you don't reach a live person!

    We thank you for your business in 2006, and wish all our customers, collaborators, distributors and partners a happy holiday, and a safe and prosperous 2007.

    More information:

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    Other Recent Publications

    Ogata and group demonstrated an alternative approach to combined fluorescent and gold labeling in their recent paper in Molecular and Cellular Biology: detecting green fluorescent protein (GFP) using a Nanogold®-labeled antibody. Eukaryotic cells deal with accumulation of unfolded proteins in the endoplasmic reticulum (ER) by the unfolded protein response: induction of molecular chaperones, translational attenuation, and ER-associated degradation. This autophagy system was found to be activated as a novel signaling pathway in response to ER stress. Treatment of SK-N-SH neuroblastoma cells with ER stressors markedly induced the formation of autophagosomes: these were recognized at the ultrastructural level by the formation of green fluorescent protein (GFP)-LC3-labeled structures (GFP-LC3 "dots"), representing autophagosomes, and was extensively induced in cells exposed to ER stress with conversion from LC3-I to LC3-II. GFP-LC3 was localized at the electron microscopic level using preembedding silver-enhanced immunogold labeling. SK-N-SH cells were cultured on coverslips and transfected with an expression vector for GFP-LC3. 30 hours after transfection, they were treated with 2 µg/mL tunicamycin for 6 hours, then fixed with 4% paraformaldehyde and 0.01% glutaraldehyde in phosphate-buffered saline (PBS) for 30 minutes, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes and blocked with 5% bovine serum albumin, 5% normal goat serum, and 0.02% NaN3 in PBS for 30 minutes. The cells were then incubated with a rat anti-GFP antibody at a dilution of 1:100 in blocking buffer for 14 hours, and after four rinses with blocking buffer over 30 minutes, with Nanogold goat anti-rat IgG at a dilution of 1:50 in blocking buffer for 18 hours. After another four rinses with PBS over 30 minutes, the cells were fixed with 1% glutaraldehyde in PBS for 15 minutes, washed and enhanced with HQ silver for 8 minutes at 20°C in the dark. Finally, specimens were washed with distilled water, postfixed in 0.1% OsO4 for 30 minutes, dehydrated through a graded ethanol series, and embedded in epoxy resin. In IRE1-deficient cells or cells treated with c-Jun N-terminal kinase (JNK) inhibitor, autophagy induced by ER stress was inhibited, indicating that the IRE1-JNK pathway is required for autophagy activation. In contrast, in PERK-deficient cells and ATF6 knockdown cells, autophagy was induced after ER stress in a manner similar to that observed in wild-type cells. Disturbance of autophagy rendered cells vulnerable to ER stress, indicating that autophagy plays important roles in cell survival after ER stress.

    Reference:

    • Ogata, M.; Hino, S.-I.; Saito, A.; Morikawa, K.; Kondo, S.; Kanemoto, S.; Murakami, T.; Taniguchi, M.; Tanii, I.; Yoshinaga, K.; Shiosaka, S.; Hammarback, J. A.; Urano, F., and Imaizumi, K.: Autophagy Is Activated for Cell Survival after Endoplasmic Reticulum Stress . Mol. Cell. Biol., 26, 9220-9231 (2006).

    Our own studies of combined fluorescent and gold probes and the interest in gold-quenched molecular beacons are two aspects of a growing interest in the enhancement of light-matter interactions near metal surfaces. Another is surface-enhanced Raman spectroscopy (SERS), which is based on the enhancement of local fields associated with excitation of collective electron oscillations in metals (surface plasmons, or SPs), which can lead to increases of orders of magnitude in molecular Raman cross-sections. Interactions with SPs can also significantly affect photoluminescence (PL) intensities of light-emitting chromophores. However, because of the competition between field enhancement and nonradiative damping due to energy transfer to SPs, the proximal metal can either enhance or decrease the PL intensity. Liu and co-workers now report the synthesis and characterization of well-defined hybrid structures comprising a gold core overcoated with a silica shell, followed by a dense monolayer of colloidal CdSe quantum dots (QDs) or semiconductor nanoparticles. A silica shell of controlled thickness was used as a dielectric spacer, providing a simple means for tuning interactions between the QDs and the metal core. By varying the silica shell thickness, the interaction was switched between PL quenching and enhancement. The synthesis included a final step, the self-assembly of QDs onto the silica shell via simple titration of the QD solution with the prefabricated silica-coated gold particles. This approach allowed accurate quantitative analysis of the effect of a metal on the QD PL intensity.

    Reference:

    • Liu, N.; Prall, B. S., and Klimov, V. I.: Hybrid gold/silica/nanocrystal-quantum-dot superstructures: synthesis and analysis of semiconductor-metal interactions. J. Am. Chem. Soc., 128, 15362-15363 (2006).

    Balazs and colleagues review nanoparticle-polymer composites in their recent article in Science. Combining polymers and nanoparticles provides methods for engineering flexible composites with novel electrical, optical, or mechanical properties, and recent advances have revealed methods to exploit both enthalpic and entropic interactions to direct the spatial distribution of nanoparticles and control the macroscopic performance of the material. Examples include tailoring the particle coating and size to create self-healing materials for improved sustainability, and self-corralling rods for photovoltaic applications. Future challenges that are discussed include the synthesis of hierarchically structured composites in which different sublayers contribute different functions to yield mechanically integrated multifunctional materials.

    Reference:

    • Balazs, A. C.; Emrick, T., and Russell, T. P.: Nanoparticle polymer composites: where two small worlds meet. Science, 314, 1107-1110 (2006).

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