N A N O P R O B E S     E - N E W S

Examples of the staining obtained using this reagent, and a comparison with DAB, are shown below. When used for in situ hybridization, EnzMet™ readily visualizes endogenous copies of single genes with almost no background. Unlike FISH, it allows accurate gene quantitation in the brightfield light microscope. This is more accessible to many users; furthermore, the signal is not subject to fading or bleaching, as many fluorophores are. In immunohistochemistry (IHC), it produces a highly resolved black signal with virtually no diffusion, allowing clear visualization of the underlying tissue morphology; this and its black color make it easy to differentiate from other stains.

Upper left: EnzMet™ detection of the amplification of individual HER2 gene copies in paraffin-embedded human invasive breast carcinoma biopsy; normal, non-amplified cells contain two copies of the HER2 gene, while the infiltrating HER2-amplified carcinoma cells show multiple copies (original magnification X 400. Image courtesy of Dr. R. R. Tubbs, Cleveland Clinic Foundation). Upper right: EnzMet™ staining of epithelial cytokeratins in paraffin-embedded human prostate adenocarcinoma using a secondary immunoperoxidase method (original magnification x 400). Lower left: Imunoperoxidase staining of epithelial cytokeratins in paraffin-embedded human bladder tumor: secondary immunoperoxidase with DAB; and Lower right: secondary immunoperoxidase method using EnzMet™ (original magnification x 400).

EnzMet™ for use in automated ISH and IHC slide staining and diagnostic applications is being commercialized by Ventana Medical Systems, under the product name SISH (Silver In Situ Hybridization. However, for manual staining procedures, you can purchase EnzMet™ for ISH and IHC from Nanoprobes. Full instructions for use are also now online.

References:

• Powell, R. D.; Pettay, J. D.; Powell, W. C.; Roche, P. C.; Grogan, T. M.; Hainfeld, J. F., and Tubbs, R. R.: Metallographic in situ hybridization. Hum. Pathol., 38, 1145-1159 (2007).
  • Abstract (courtesy of Science Direct).
• Tubbs R.; Pettay J.; Powell R.; Hicks D. G.; Roche P.; Powell W.; Grogan T., and Hainfeld, J. F.: High-resolution immunophenotyping of subcellular compartments in tissue microarrays by enzyme metallography. Appl. Immunohistochem. Mol. Morphol., 13, 371-375 (2005).
  • Abstract (courtesy of the PubMed (Medline)).

Western and Protein Blotting

EnzMet™ is also one of the cleanest and most sensitive detection reagents we have tried for Western blots. A comparison between conventional DAB development and EnzMet™ development on a Western blot is shown below:

Comparison of Western blot detection of his-tagged Fusion Protein using HRP conjugates developed with DAB (Panel A) and EnzMet™ (Panel B). After transfer, membranes were incubated with anti-His-Tag (6xHis) monoclonal antibody, followed by BSA blocking, and then exposed to Horseradish peroxidase (HRP)-conjugated secondary antibody. The His-tagged fusion proteins were then visualized by DAB detection (Panel A) or EnzMet detection (Panel B). Lanes 1 and 4: 0.1 µg of 34 kDa his-tagged ATF-1. Lanes 2 and 5: 0.1 µg of 68 kDa his-tagged YY1. Lanes 3 and 6: 0.1 µg BSA and 0.1 µg ovalbumin.

In most cases, the EnzMet™ protocol using EnzMet™ for Blots below may be substituted for conventional DAB development without further modification of the protocol. However, because of its greater sensitivity, greater dilutions of either primary antibody or secondary probes may be required to achieve the optimum combination of sensitivity and clarity. A five-fold to ten-fold additional dilution has been found to give good results in immunohistochemical experiments and is likely to be appropriate here also.

Protocol:

1. Wash with buffer containing 0.1% Tween-20 for 3 x 5 minutes.
   Note: Phosphate buffered saline, tris buffered saline or other wash buffers can be used. Including 0.1 % (w/v) Tween-20 in the wash buffer was found to be helpful in reducing non-specific binding.

2. Wash with deionized water for 3 x 5 minutes.

3. Shake off excess water. Cover membrane with 6 mL (or 3 volumes) of EnzMet™ Detect A. Incubate for 4 minutes.
   Note: Excess water can lead to the dilution of EnzMet™ reagents, resulting in weak staining and results which are difficult of reproducing.

4. Add 2 mL (or 1 volume) of EnzMet™ Detect B to the membrane, and gently mix Solutions A and B. Incubate for 4 minutes.

5. Add 2 mL (or 1 volume) of EnzMet™ Detect C to the membrane, and gently mix Solutions A, B and C. Incubate for 9 - 25 minutes, or until satisfactory staining is achieved.
   Note: The EnzMet™ incubation time mainly depends on the target concentrations and staining temperature. Longer incubation may be needed for visualizing low concentration targets. However, longer incubation may lead to some non specific background staining. The variation of EnzMet™ staining temperature can affect its silver deposition rate. Lower temperature slows down the deposition process, and thus a longer staining time may be required to reach a certain degree of staining density and sensitivity.

6. Wash with deionized water for 3 x 5 minutes.

7. Air dry membrane for record.

You can purchase EnzMet™ for blotting from Nanoprobes; instructions and protocols for use are also now available online.

Reference:

• Liu, W.; Mitra, D.; Powell, R.; Tubbs, R.; Pettay, J., and Hainfeld, J.: Enzyme Metallography Silver Deposition for HRP Detection. Presented at ASCB 2007, Washington DC, December 1-4, 2007,: Poster # B349, Presentation # 1209.

Electron Microscopy

But we don't wish to leave out electron microscopists. The granular IHC staining shows that the reaction product essentially does not diffuse, providing the high resolution necessary for electron microscopy. Here, it has the great advantage that the small size of the enzymatic probe and in situ metal deposition can afford improved access to interior or to hindered antigens, and provide denser labeling than larger colloidal gold probes, while the high contrast means the deposited silver is readily visualized. At the Microscopy and Microanalysis 2006 meeting, we presented the results of a detailed study, conducted in collaboration with the Department of Biological Sciences at Rutgers University, in which EnzMet™ was used to localize polar tube proteins in Microsporida at the light and electron microscopic level. Microsporida are parasitic organisms that are important opportunistic pathogens in AIDS and other immune compromised patients. They are responsible for chronic diarrhea, malabsorption syndromes, myositis, and disseminating infections demonstrated in all tissues of the body. Approximately a dozen different microsporidia infect humans: all form a diagnostic spore containing a coiled polar filament surrounding the single nucleus or paired abutted nuclei (diplokaryon) and its associated cytoplasmic organelles, the sporoplasm; upon germination, the polar filament is everted to become a tubule, through which the spore contents travel to become the infective sporoplasm.

Cultured RK-13 cells infected with E. hellem microsporidia were grown on slides, immunofixed (for electron microscopy), and stored in PBS buffer. These were incubated with primary antibody (anti polar tube – PTP-55 [6], 1:100) for one hour. A universal detection system incorporating a biotinylated secondary antibody and polymerized peroxidase-streptavidin detection (I-View, Ventana Medical Systems) was applied. After washing with PBS-0.01% Tween-20, distilled water, and 0.02 M sodium citrate buffer at pH 3.8, specimens were developed with a modified formulation of the enzyme metallographic reagent (Nanoprobes, Incorporated), washed again with 0.02 M sodium citrate buffer at pH 3.8, rinsed with deionized water, and coverslipped. After light microscope examination, areas of interest were marked on the back of the slides. The cover glasses were removed, and the slides rinsed in distilled water, dehydrated through a series of ethanol solutions (50% - 100%) and infiltrated with Araldite 502 resin (EMS, PA) overnight. Marked areas were covered with BEEM capsules filled with resin, and embedded at 60°C for 24 hours. Thin sections were cut, placed on copper grids, and stained with uranyl acetate and lead citrate. Samples were examined using a transmission electron microscope. Some results are shown below.

Upper left: Schematic showing the mode of action of enzyme metallography. Micrographs show RK-13 cells infected with E. hellem microsporidia, stained using anti-polar tube antibody (PTP-55) and I-View universal peroxidase detection system (Ventana Medical Systems), developed with enzyme metallography. (a) Brightfield light microscopy with 40X dry objective; (b) Brightfield with 40X oil immersion objective, showing infective sporoplasm (dark arrow) and empty spore (white arrow); (c) TEM: staining with primary antibody and enzyme metallography development, showing heavily decorated polar tube, empty spore (dark arrow) and infective sporoplasm (white arrow). (d) control with primary antibody omitted, showing counterstained polar tube.

The polar tubes were easily observed with brightfield optics, and background was very clean: almost no non-specific deposits were observed on the cells and surrounding matrix. The enzyme metallography visualization is more sensitive, with lower background and higher resolution than DAB staining, and for the first time, brings sufficient staining intensity for diagnostic identification to light microscopy, providing a robust and practical method for diagnosis in less developed regions of the world where microsporida are a major public health concern and where electron microscopy, the usual diagnostic tool, is not available. At the same time, the electron microscopy results show that the method also provides superior research tool for ultrastructural examination. These results were obtained using our EnzMet™ HRP Detection Kit for Research Applications (catalog # 6010); we plan to introduce a new formulation optimized for electron microscopy in future.

Reference:

• Powell, R.; Joshi, V.; Thelian, A.; Liu, W.; Takvorian, P.; Cali, A., and Hainfeld, J.: Light and Electron Microscopy of Microsporida using Enzyme Metallography. Microsc. Microanal., 12, (Suppl. 2: Proceedings); Kotula, P.; Marko, M.; Scott, J.-H.; Gauvin, R.; Beniac, D.; Lucas, G.; McKernan, S., and Shields, J. (Eds.), Cambridge University Press, New York, NY, 424CD (2006).
  • Complete paper from Microscopy and Microanalysis 2006

Conductive Array Biochips

In addition to its use for staining and blotting, EnzMet™ may also be used for highly specific electrical detection using conductive array biochips: enzyme-labeled DNA probes were developed with EnzMet™ to form conductive bridges after binding to targets patterned to connect two electrodes. If you wish to try this procedure, you should use our third EnzMet™ product, EnzMet™ HRP Detection Kit for Research Applications (catalog # 6010). Instructions are available online.

Reference:

• Moller, R.; Powell, R. D.; Hainfeld, J. F., and Fritzsche, W.: Enzymatic control of metal deposition as key step for a low-background electrical detection for DNA chips. Nano Lett., 5, 1475-1482 (2005).
  • Abstract (courtesy of ACS Publications).

More information:

• EnzMet™: catalog and general information
• HRP Detection Kit for IHC/ISH: Product information and instructions
• EnzMet™ Western Blot HRP Detection Kit: Product information and instructions
• EnzMet™ HRP Detection Kit for Research Applications: Product information and instructions
• EnzMet™: Applications
• EnzMet™: References
• Enzyme Metallography: A Simple New Staining Method (our paper from Microscopy & Microanalysis 2002

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Gold Enhancement: Better than Silver?

Enhancement of Nanogold® by GoldEnhance™: mechanism. Final particle size is controlled by enhancement time.

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.

Left: Pre-embedding immunolabeling using Nanogold-Fab’ and GoldEnhance™ EM, showing uniform enlarged particles. Center and right: DAB vs. GoldEnhance-Nanogold: Formalin-fixed serial paraffin sections of cervical carcinoma, in situ hybridized for HPV-16/18 using a biotinylated probe (bar = 10?m). (center) DAB-peroxidase; (right) Nanogold-streptavidin with GoldEnhance™ (courtesy of G. W. Hacker, Medical Research Coordination Center, University of Salzburg).

References:

• Beznoussenko, G. V.; Dolgikh, V. V.; Seliverstova, E. V.; Semenov, P. B.; Tokarev, Y. S.; Trucco, A.; Micaroni, M.; Di Giandomenico D.; Auinger, P.; Senderskiy, I. V.; Skarlato, S. O.; Snigirevskaya, E. S.; Komissarchik, Y. Y.; Pavelka, M.; De Matteis, M. A.; Luini, A.; Sokolova, Y. Y., and Mironov, A. A.: Analogs of the Golgi complex in microsporidia: structure and avesicular mechanisms of function. J. Cell Sci., 120, 1288-1298 (2007).
  • Abstract (courtesy of the Journal of Cell Science).
  • Reprint (PDF) (courtesy of the Journal of Cell Science).
• Graf, A. H.; Cheung, A. L.; Hauser-Kornberger, C.; Dandachi, N.; Tubbs, R. R.; Dietze, O., and Hacker, G. W.: Clinical relevance of HPV 16/18 testing methods in cervical squamous cell carcinoma. Appl. Immunohistochem. Molecul. Morphol., 8, 300-309 (2000).
  • Abstract (courtesy of the Applied Immunohistochemistry & Molecular Morphology).

If you have been using Nanogold®: for detection on nitrocellulose membrane blots (immunodot blots or Westerns) and experienced less than ideal detection, we have developed an optimized detection procedure that maintains the already very high sensitivity, but combines it with a greatly reduced background and enhanced signal clarity. For the best results, we recommend using a procedure that incorporates the following components:

• Use gold enhancement rather than silver enhancement to develop the signal after application of the Nanogold conjugate. In most cases, this will provide substantially lower background or non-specific signal, while maintaining a similar level of sensitivity to silver enhancement.

• Incorporate 0.1% Tween-20 (detergent) in the buffers used for blocking, antibody incubation, and washing. This will dramatically reduce background binding.

• Include 1% nonfat dried milk (you can use the material sold in supermarkets and food stores) as an additive in the incubation buffer (the buffer in which the Nanogold is dissolved and applied to the blot) and 5% nonfat dried milk in the blocking buffer used to block the membrane before application of antibodies.

Suggested procedure:

Reagents and Equipment:

• Phosphate buffered saline (PBS): 20 mM sodium phosphate buffer pH 7.4 and 150 mM NaCl.
• Specific antigen (target protein or other biomolecule).
• Nitrocellulose (NC) membrane 0.2 µm pore size.
• Blotting Paper to wick membrane dry.
• Orbital Shaker
• Washing buffer (TBS-Tween 20): 20 mM Tris pH 7.6, with 150 mM NaCl and 0.1 % Tween-20.
• Nonfat dried milk (Carnation)
• GoldEnhance EM (Nanoprobes Product No. 2113).
• Specific Nanogold antibody conjugate.

Procedure:

Antigen Application:

1. Prepare antigen solutions with a series of dilutions (0.01mg/mL, 0.001mg/mL, 0.0005mg/mL, 0.0001 mg/mL, 0.00005 mg/mL, 0.00001 mg/mL and 0.000005 mg/mL) using PBS, pH7.4.
2. Pipette 1 µL of above solutions to a dry nitrocellulose membrane; prepare 2 duplicates as a negative control.
   • Negative control 1: No antigen, No antibody.
   • Negative control 2: No antigen with NG-conjugate incubation.

3. Air-dry for 30 minutes

Blocking:

1. Immerse membranes in 8 mL of TBS-Tween 20 for 5 minutes.
2. Block membranes in 8 mL of TBS-Tween 20 containing 5 % nonfat dried milk for 30 minutes at room temperature.

Binding of Nanogold antibody conjugate:

1. Dilute Nanogold antibody conjugate in TBS-Tween20 containing 1% nonfat dried milk to 4 µg/mL (1:20 Dilution: 300 µL conjugate + 5.30 ml TBS-gelatin containing 1% nonfat dried milk).
2. Incubate the membranes in 8 mL of diluted conjugate solution for 30 minutes at room temperature.
3. Incubate the control membrane in 8 mL of TBS-Tween20 containing 1% nonfat dried milk for 30 minutes at room temperature.

Autometallographic Detection:

1. Wash membranes three times for 3 min each in 8 mL of TBS-Tween 20. Wash membranes thoroughly in 8 mL of deionized water (4 x 3 minutes). Make sure strips are washed separately according to what they are incubated in (strips incubated in one lot of a conjugate are washed in a separate dish from strips that are incubated in TBS-Tween 20 with 1% nonfat dried milk without conjugate, strips incubated in different lots are washed separately).
2. Perform Gold Enhancement according to instructions (mix solutions A and B, wait 5 minutes, then add C and D).
3. Record the number of observed spots and time when the spots appear. Record the time when background appears on the control membrane.
4. After 15 minutes, the enhancement solution is removed. Rinse membranes with water (3 x 3 minutes) and air-dry for storage.

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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The Secret of Extreme Radiotolerance, Exposed with Nano-W

Wild-type and DdrA_(1–160) were overproduced in E. coli using p12713, with an additional stop point inserted for the truncated protein. This plasmid was constructed by cloning a NdeI/BamHI DNA fragment, corresponding to the D. deserti ddrA sequence (generated by PCR with the FV41/FV43 primers and D. deserti genomic DNA as template) between the NdeI/BamHI sites of pET-TEVDdrA(1–160) was carried out by elution of the cellular extract from Ni-NTA beads by 0.15 M Imidazole, 1 M NaCl and 0.05 M Tris–HCl, pH 8.0. The purest fractions were pooled and concentrated with a large cutoff (100 kDa) centricon, then after mass spectroscopy, a final gel filtration chromatography step was added to the purification protocol using a Superdex 200 HiLoad 16/60 column pre-equilibrated in 0.15 M NaCl, 0.02 M Tris–HCl, pH 7.5): a single peak was observed. The secondary structures and linker region predictions of D. deserti DdrA were performed with the Jpred and DPL programs. SDS-PAGE gel analysis of the elution peak obtained with the truncated protein after the gel filtration indicated one single major band at 20 kDa and several minor bands corresponding to higher molecular weight species of DdrA_(1–160).

For electron microscopy, different fractions of the peak eluted from the gel filtration column were collected and observed separately with negative staining, a method used for high-resolution electron microscopy to define the edges of particulate or suspended specimens with low contrast. The sample was applied to the clean side of a thin carbon film on carbon–mica interface, and the carbon film with the absorbed sample was floated on a drop of Nano-W solution. A 400-mesh copper grid was put on top of the floating carbon film, then the whole was turned upside down and used to catch a second layer of carbon film floating on another drop of NanoW. In this way, an entirely and uniformly stained sample was trapped between two thin layers of carbon. The grids were observed under low-dose conditions with a tungsten filament at 100 kV.

Our negative stains, NanoVan (vanadate) and Nano-W (tungstate) are building a solid volume of publications, are ideal for this type of work because they have a highly amorphous structure and fine grain which provides for maximum resolution in specimen imaging, since crystallization can obscure features of interest. In addition, NanoVan is ideal for use with smaller gold labels such as Nanogold®: because the stain is less electron-dense than other negative stains such as uranyl acetate or lead citrate, contrast between the gold particles and their environment is preserved. It is very fine-grained and highly amorphous, and has been used for a number of high-resolution STEM and TEM studies of virus and protein ultrastructure. Nano-W gives a more dense stain, and is more suited to use with larger gold labels. NanoVan and Nano-W are based on organic salts of vanadium and tungsten respectively.

Advantages of these reagents:

• NanoVan and Nano-W are completely miscible: they may be mixed in different proportions to give any desired intermediate stain density.
• Near-neutral pH results in better ultrastructural preservation.
• NanoVan is less susceptible to electron beam damage than uranyl acetate.
• Fine grain allows high imaging resolution.

Schematic showing how negative stains work (left) and high-resolution electron micrographs obtained using a scanning transmission electron microscope. (a) Tobacco Mosaic Virus (TMV) negatively stained with 2 % uranyl acetate; (b) TMV stained with 1 % methylamine vanadate (NanoVan); both samples imaged with a dose of 104 eI/nm2. Original full width 128 nm for each image. (c) Side view of groEL (large arrow) labeled with 1.4 nm gold cluster (Nanogold, small arrow) imaged in methylamine vanadate. Note clear visibility of subunit structure and gold cluster. Full width 128 nm. Specimen kindly provided by A. Horwich, Yale University.

Visual inspection indicated that all the fractions contained a small ring-shaped species, together with some larger species. The most distal fraction seemed the most homogeneous, and analysis was focused on this particular fraction. Images were recorded on film at a nominal magnification of 40 000 x, and the negatives digitized on a Zeiss SCAI scanner at a pixel size of 7 ?m, corresponding to 1.75 Å/pixel at the specimen level. Image processing was performed with IMAGIC and EMAN software on 15,166 subframes containing individual DdrA(1–160) particles selected interactively from micrographs at different defoci to better fill the zeroes of the contrast transfer function. Multivariate statistical analysis (MSA) and classification of the aligned data set allowed separation of the big species from the small ring-shaped species, and demonstrated clearly the 3-fold rotational symmetry of the large DdrA_(1–160) oligomer. The characteristic class averages of the large oligomer were then used as a set of references for multi-reference alignment (MRA) followed by MSA, classification, and refinement to an estimated resolution of the reconstruction of about 23 Å according to the 0.5 threshold. A fit of the crystal structure of the homologous-pairing domain of human Rad52 protein into the electron microscopy reconstruction was done with the UROX software; Only the N-terminal domain residues ranged from 25 to 178 of Rad52 were considered for the fitting. The last C-terminal residues from 179 to 209 were excluded because they include a loop region in the crystal structure. The buried accessible surface was estimated by AREAIMOL from the CCP4 package.

Two types of assemblies could be detected immediately from the negative stain images: smaller ring-shaped species of 7.5 nm in diameter, and larger ones about 13.5 nm in size. The latter were randomly oriented on the grid, and some of their projections showed that the largest objects were composed of rings of the same dimensions as the small particles. Therefore, it was inferred that the small ring-shaped species constituted the building blocks of the large DdrA_{(1–160) complex}. Although it is not functional in vivo, the truncated DdrA protein keeps its DNA binding ability at the wild-type level. DdrA(1–160) has a complex three-dimensional structure, based on a heptameric ring that can self-associate to form a larger molecular weight assembly. The authors conclude that this complex architecture may play a role in the substrate specificity, and favors an efficient DNA repair. In the cell, the large assemblies could correspond to a transient state occurring during the DNA repair to put together the repair machinery, including several DNA single strands and other unknown cellular partners. A complex pattern of self-association has also described for the wild type and truncated forms of Rad52, and it was suggested that this is important to promote DNA end-joining. This type of large, complex organization with conformational flexibility would function to optimize the interactions between these different molecular elements.

Reference:

• Gutsche, I.; Vujicic-Zagar, A.; Siebert, X.; Servant, P.; Vannier, F.; Castaing, B.; Gallet, B.; Heulin T.; de Groot, A.; Sommer, S., and Serre, L.: Complex oligomeric structure of a truncated form of DdrA: A protein required for the extreme radiotolerance of Deinococcus. Biochim. Biophys. Acta, 1784, 1050-1058 (2008).
  • Abstract (courtesy of Science Direct).

More information:

• Catalog information: Negative stain reagents
• NanoVan: Product information and instructions
• Nano-W: Product information and instructions
• Technical help: Negative staining
• NanoVan Negative stain: Our paper from Microscopy and Microanalysis 1994
• Negative staining: References

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Nanogold® and Addiction

• High specimen penetration.
• Highest resolution - the gold is nearer to the target.
• High labeling density providing more quantitative labeling than other immunogold probes.
• High level of access even to nuclear, restricted or hindered antigens.

Upper: Size comparison of Nanogold-Fab' with conventional 5 nm colloidal gold-IgG probe, showing overall probe size and distance of gold from target. lower left: Scanning transmission electron micrograph of Nanogold-labeled Fab', showing attachment of the Nanogold at the hinge region of the Fab' (image width 86 nm). lower right: Nanogold®-Fab' goat anti-rabbit IgG labeling the K+ channel Kv2.1 subunit in rat brain, followed by HQ Silver (Catalog # 2012) enhancement. Note high density and specificity of immunostaining, even elucidating subunit localization to cytoplasmic side of cell membrane and outer stacks of the Golgi; axons and terminals are clearly negative. Work done by J. Du, J.-H. Tao-Cheng, P. Zerfas, and C. J. McBain, NIH. See Neuroscience, 84, 37-48 (1998). Bar = 1 micron.

Male Sprague–Dawley rats were subjected to a chronic cocaine exposure cocaine administration regimen routinely used to induce behavioral sensitization to psychostimulants in rats. For perfusion, all animals were deeply anesthetized with a cocktail of ketamine (60–100 mg/kg, i.p.) and dormitor (0.1 mg/kg, i.p.), then transcardially perfused with cold oxygenated Ringer’s solution followed by a fixative containing 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Brains were removed, post-fixed in 4% paraformaldehyde for 24 h, cut into 60-µm-thick sections using a vibrating microtome and stored in phosphate-buffered saline (PBS; 0.01 M, pH 7.4) at 4°C until used. Before immunocytochemical reactions, all sections were treated with 1% sodium borohydride solution for 20 minutes, then washed with phosphate-buffered saline (PBS). sections were incubated for 30 min in PBS containing 5% dry milk at RT and then rinsed in TBS-gelatin buffer (0.02 M and pH 7.6).

For electron microscopic labeling, sections were then incubated with primary antibody solutions with 1% dry milk in TBS-gelatin buffer for 24 hours at room temperature (RT), rinsed in TBS-gelatin, then treated for two hours at RT with secondary goat Nanogold-IgG anti-rabbit IgG diluted 1:100 in 1% dry milk in TBS-gelatin. Sections were then rinsed in TBS-gelatin and 2% sodium acetate buffer, after which the Nanogold particles were silver intensified to 30–50 nm using HQ silver for approximately 10 minutes. The tissue was then rinsed in phosphate buffer (0.1 M, pH 7.4) and treated with 0.5% OsO₄ for 10 minutes, then returned to PB and dehydrated with increasing concentrations of ethanol. At 70% ETOH, 1% uranyl acetate was added to the solution for 10 minutes to increase the contrast of the tissue in the electron microscope. Following dehydration, sections were treated with propylene oxide and embedded in epoxy resin for 12 hours, mounted onto slides and placed in a 60°C oven for 48 hours. Separate samples of the nucleus accumbens core and medial shell were cut out of the larger sections, mounted onto resin blocks and cut into 60-nm sections using an ultramicrotome; the 60-nm sections were collected on Pioloform-coated copper grids, stained with lead citrate for 5 minutes to enhance tissue contrast, then examined on electron microscope. Electron micrographs were taken and saved with a CCD camera.

After a single cocaine injection (30 mg/kg) and 45 minute withdrawal, there was a significant decrease in the proportion of plasma membrane–bound mGluR1a in accumbens shell dendrites. Similarly, the proportion of plasma membrane– bound mGluR1a was decreased in large dendrites of accumbens core neurons following chronic cocaine exposure (i.e. 1-week treatment followed by 3-week withdrawal). However, neither acute nor chronic cocaine treatments induced significant change in the localization of mGluR5 in accumbens core and shell, which is in contrast with the significant reduction of plasma membrane–bound mGluR1a and mGluR5 induced by local intraaccumbens administration of the group I mGluR agonist, (RS)-3,5-dihydroxyphenylglycine (DHPG). In conclusion, these findings demonstrate that cocaine-induced glutamate imbalance has modest effects on the trafficking of group I mGluRs in the nucleus accumbens. These results provide valuable information on the neuroadaptive mechanisms of accumbens group I mGluRs in response to cocaine administration.

Reference:

• Mitrano, D. A.; Arnold, C., and Smith, Y.: Subcellular and subsynaptic localization of group I metabotropic glutamate receptors in the nucleus accumbens of cocaine-treated rats. Neuroscience, 154, 653-666.
  • Abstract (courtesy of Science Direct).

More information:

• Special Feature: Nanogold pre-embedding immunoelectron microscopy
• Nanogold conjugates: catalog information
• Nanogold conjugates: product information and instructions
• References: Nanogold for pre-embedding immunolabeling
• Technical help: Nanogold conjugates

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Nanoprobes at Microscopy and Microanalysis 2008

Please note that Nanoprobes will be closed on Thursday, July 3 and Friday, July 4 in observance of the United States Independence Day holiday. Correspondence will be answered and pending orders received during the holiday will be shipped on Monday, July 7 or as soon as possible thereafter.

More information:

• Microscopy & Microanalysis 2008
• Microscopy & Microanalysis 2008 - Program search engine
• Meeting information pamphlet and symposium timetable.
• Nanoprobes news and press releases

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Other Recent Publications

Reference:

• Patterson, G. H.; Hirschberg, K.; Polishchuk, R. S.; Gerlich, D.; Phair, R. D, and Lippincott-Schwartz, J.: Transport through the Golgi apparatus by rapid partitioning within a two-phase membrane system. Cell, 133, 1055-1067 (2008).
  • Summary (courtesy of Cell).

Reference:

• He, M.; Li, K.; Xiao, J., and Zhou, Y.: Rapid bio-barcode assay for multiplex DNA detection based on capillary DNA Analyzer. J. Virol. Methods, 151, 126-131 (2008).
  • Abstract (courtesy of Science Direct).

Gautier and Bürgi shed light on the role of thiol ligands in determining the structure and exchange kinetics of small thiol-stabilized gold nanoparticles containing a few tens of gold atoms, in their recent paper in the Journal of the American Chemical Society. The thiolate-for-thiolate ligand exchange was performed on well-defined gold nanoparticles under an inert atmosphere, without any modification of the core size. This reaction is faster than the well-known core etching process. Unexpectedly, if a chiral thiol is exchanged for its opposite enantiomer, the optical activity in the metal-based electronic transitions is reversed, although the form of the circular dichroism (CD) spectrum remains largely unchanged. The extent of inversion corresponds to the overall enantiomeric excess of the chiral ligand in the system. This shows that the chiral arrangement of metal atoms on the metal particle (surface) cannot withstand the driving force imposed by the ligand of opposite absolute configuration. If the incoming thiol has a different structure, the electronic transitions in the metal core are slightly modified whereas the absorption onset remains unchanged. These results emphasize the influence of the thiols on the structure of the gold nanoparticles, and can be used to give insight into the ligand exchange pathways.

Reference:

• Gautier, C., and Bürgi, T.: Chiral inversion of gold nanoparticles. J. Amer. Chem. Soc., 130, 7077-7084 (2008).
  • Abstract (courtesy of ACS Publications).

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