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Updated: January 7, 2003

N A N O P R O B E S     E - N E W S

Vol. 4, No. 1          January 7, 2003


This monthly newsletter is to keep you informed about techniques to improve your immunogold labeling, highlight interesting articles and novel metal nanoparticle applications, and answer your questions. We hope you enjoy it and find it useful.

Have questions, or issues you would like to see addressed in the next issue? Let us know by e-mailing [email protected].

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Nanogold® Labeling shows how Tau binds To Microtubules

In July 2002 we reported the work of Al-Bassam and co-workers, who used undecagold to localize the site of microtubule-associated protein 2 (MAP2) and tau protein binding on the surface of pre-assembled microtubule protofilaments; Cryo-EM and helical image analysis showed that both the IR and MAP2 elements lie along the exterior ridges of microtubules. 1999). Amos and co-workers have now used tau protein, labeled with Nanogold® at a repeat motif in the microtubule-binding domain, to study tau binding during microtubule assembly. Three-dimensional electron cryomicroscopy indicates that a repeat motif occupies a similar site to taxol on the inner surface of the microtubules; this was confirmed by pelleting assays showing that when tubulin and tau are coassembled into microtubules, the presence of taxol reduces the amount of tau incorporated. The authors conclude that one of the tau repeat loops is the natural substrate that occupies to the taxol-binding pocket in beta-tubulin.

Human 4R-tau with mutations to replace Cys291 and Cys322 with isoleucine, and Ser305 with cysteine, was labeled with Monomaleimido-Nanogold reagent; labeled tau was separated from an excess Nanogold on a Superdex-200 column. UV/visible absorption measurements at 280 and 420nm indicated that 93 - 97% of the tau molecules were labeled. After separation from free gold, the labelled protein was incorporated immediately into microtubules. Tau from the 97%-labeled fraction was incubated with tubulin to final concentrations of 1.0 mg/ml tubulin, 0.3 mg/ml tau, 1 mM GMPCPP, 0.1 M TMAO and 5% DMSO at 37°C for 20 min. For some samples, 10 mM kinesin (rat KD340) was added with 1 mM AMPPNP. Cryogrids were made and examined on a Philips CM-12, 120 KeV cryomicroscope at 45 0003 magnification and 1.25 mm underfocus. Micrographs were scanned in 28 mm steps and reconstructed using the MRC-LMB software package. Layer-line data from different images, to a resolution of ~3.5 nm, were compared and those that were in best agreement were averaged (at least 6000 nm of 15-pf microtubule, 12 000 tubulin dimers, 3000 tau molecules).

Reference:

Kar, S.; Fan, J.; Smith, M. J.; Goedert, M., and Amos, L. A.: Repeat motifs of tau bind to the insides of microtubules in the absence of taxol. EMBO J., 22, 70-77. (2003).

Abstract (courtesy of the EMBO Journal):
http://emboj.oupjournals.org/cgi/content/abstract/22/1/70

More information:

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Combined Alexa Fluor®* and Nanogold® Probes

Alexa Fluor® 488 and 594 FluoroNanogold probes are now available. These new, brighter combined fluorescent and gold probes offer new performance levels and additional features:
  • Increased fluorescence brightness and higher quantum yield.
  • Improved solubility means lower background and higher signal-to-noise ratios.
  • Fluorescence remains high and consistent across wider pH range.
  • Uses fluorescein (Alexa Fluor® 488) or Texas Red (Alexa Fluor® 594) filter sets.
  • Available in 1 mL or affordable 0.5 mL sizes.
With Alexa Fluor® 594, you can differentiate a FluoroNanogold-labeled target from a second target labeled with fluorescein, Alexa Fluor® 488, green fluorescent protein, or other fluorophores.

Visit the FluoroNanogold section of our online catalog for new images demonstrating the sensitivity of this probe, and the correlation now possible between fluorescence and electron microscopy; in addition, complete instructions and protocols optimized for these new probes are available in the product information section of our site.

More information:

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

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Two Methods for Nanogold® Labeling of Lipid-Associated Features

The stable, water-soluble, yet non-ionizing nature of the Nanogold® surface gives it great versatility for labeling different types of targets in different environments. Nanogold-labeled lipids, in which the 1.4 nm Nanogold cluster is chemically linked to palmitoyl or dipalmitoyl phosphatidyl ethanolamine moieties, insert into liposomes, labeling them for light or electron microscope observation. One successful application of these probes is light and electron microscopic tracking of liposomal delivery drug delivery. Nanogold-labeled liposomes containing an anti-fungal channel-forming drug were shown to pass through the cell wall and enter the cytoplasm, while those without the drug did not. Reference:

Adler-Moore, J.: AmBisome targeting to fungal infections. Bone Marrow Transplantation, 14, S3-S7 (1994).

Abstract (Medline):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=7703928&dopt=Abstract

Labeling with DPPE-Nanogold has also been used to demonstrate the targeting of cationic liposomes to endothelial cells in tumors and chronic inflammation. Reference:

Thurston, G., McLean, J. W., Rizen, M., Baluk, P., Haskell, A., Murphy, T. J., Hanahan, D., and McDonald, D. M.: Cationic Liposomes Target Angiogenic Endothelial Cells in Tumors and Chronic Inflammation in Mice. J. Clin. Invest., 101, 1401-13 (1998).

Abstract (courtesy of the Journal of Clinical Investigation):
http://www.jci.org/cgi/content/abstract/101/7/1401
Reprint (courtesy of the Journal of Clinical Investigation):
http://www.jci.org/cgi/reprint/101/7/1401.pdf

The ability of gold lipids to self-organize in a regular manner and form ordered arrays makes them possible precursors to nanostructured materials. In our 1999 paper, we described the formation and morphology of gold-labeled liposomes. Brown and co-workers have described the formation of Langmuir monolayers of palmitoyl Nanogold, created by depositing an alcohol solution of nanoparticles onto deionized water at room temperature (23°C) in a Joyce-Loebl Langmuir trough. Transfer of the films to transmission electron microscope grids by the Langmuir-Schaeffer method resulted in observation of highly ordered hexagonal arrays larger than 250 x 350 nm2.

References:

  • Hainfeld, J. F.; Furuya, F. R., and Powell, R. D.: Metallosomes. J. Struct. Biol., 127, 152-160 (1999).

    Abstract (courtesy of the Journal of Structural Biology):
    Link

  • Brown, J. J.; Porter, J. A.; Daghlian, C. P., and Gibson, U. J.: Ordered Arrays of Amphiphilic Gold Nanoparticles in Langmuir Monolayers. Langmuir, 17, 7966 (2001).
More information:

Meanwhile, Heid and co-workers demonstrate dense labeling of a lipid-associated protein, adipophilin, using more conventional immunoelectron microscopy with a Nanogold-labeled secondary antibody. Endothelial cells from human umbilical cord veins, grown on coverslips, were fixed with 2% formaldehyde (freshly prepared from Paraformaldehyde) in PBS and 3% sucrose for 10 min, then permeabilized with 0.03% saponin in PBS for 5 min, and rinsed with PBS containing 0.005% saponin. Labeling was performed using hybridoma supernatant containing mab AP125 as the primary antibody (incubation for 2 h) followed by secondary anti-mouse IgG coupled to Nanogold, which was were allowed to react for 3 h. The Nanogold particles were visualized by silver enhancement, and the cells on the coverslips were then dehydrated and flat-embedded.

Reference:

Heid, H. W.; Moll, R, Schwetlick, I.; Rackwitz, H. R.; and Keenan, T. W.: Adipophilin is a specific marker of lipid accumulation in diverse cell types and diseases. Cell Tissue Res., 294, 309-21 (1998).

Abstract (courtesy of Cell and Tissue Research):
http://link.springer-ny.com/link/service/journals/00441/bibs/8294002/82940309.htm

More information:

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Obtaining the Best Silver Enhancement Results

Silver enhancement is a well-characterized procedure, and protocols for use with our HQ Silver enhancement reagent have been carefully optimized. Dr. Susan Cheng and co-workers at NINDS have refined their pre-embedding procedure for the most uniform, consistent and reliable pre-embedding labeling, using Nanogold®-Fab' and HQ Silver. Their procedure is described in full on our web Site:

Application note - pre-embedding immunoEM: www.nanoprobes.com/App3.html

References:

Nechushtan and co-workers used a similar procedure to identify a novel step in the Bax proapoptotic mechanism, and also demonstrated an alternative approach to correlative confocal fluorescent and gold labeling by targeting GFP variant fusions of the target proteins. NeBax, a member of the Bcl-2 family of proteins known to regulate mitochondria-dependent programmed cell death, was identified; Nanogold labeling enabled the macromolecular localization of Bax and Bak during cell death and their differentiation from that of fellow Bcl-2 family members Bid and Bad, and elucidation of their role. Samples were fixed with 2% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer at pH 7.4 for 30 min, washed, permeabilized, and incubated with either antiuniversal Bax 6A7 or antihuman Bax 1F6 monoclonal antibodies, both of which recognize monkey Bax, or for GFP variants, 3E6 anti-AFP. Treatment of samples with the secondary Nanogold-antibody conjugate was followed by washing and silver enhancement (HQ Silver), treatment with 0.2% OsO4, dehydration and Epoxy resin embedding.

Reference:

Nechushtan, A.; Smith, C. L.; Lamensdorf, I.; Yoon, S. H., and Youle, R. J.: Bax and Bak coalesce into novel mitochondria-associated clusters during apoptosis. J. Cell Biol., 153, 1265-76 (2001).

Abstract (courtesy of the Journal of Cell Biology):
http://www.jcb.org/cgi/content/abstract/153/6/1265 Reprint (courtesy of the Journal of Cell Biology):
http://www.jcb.org/cgi/reprint/153/6/1265

The following steps will help ensure a clean silver enhancement reaction:

  • Ensure that all samples are thoroughly washed with ultrapure or deionized water before silver enhancement: halide anions will react to form a precipitate, which is visible in the EM and will nucleate additional background staining.

  • Wash samples with a chelating agent before silver enhancement, such as 0.02 M sodium citrate buffer, pH 7.0 (with Danscher silver enhancer), 0.02 M sodium citrate buffer at pH 3.5 (with HQ Silver) or disodium EDTA (0.05 M, pH 4.6) as the last wash before adding the silver enhancement reagent.

  • Clean glassware (for mixing reagents, handling specimens or storing silver enhancers) with Farmer's solution for at least 20-30 minutes. Farmer's solution consists of 9 parts of 10 % sodium thiosulfate with 1 part of 10 % potassium ferricyanide.

  • Use plastic or teflonized (not metal) forceps and tools for handling specimens.
Additional suggestions for getting the best out of silver enhancement, as well as references, are given in the technical help section of our web site:

Technical Help - Silver enhancement: www.nanoprobes.com/TechSE.html

Related information:

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Other Recent Publications

Laboux and group have investigated postembedding immunogold labeling protocols for undecalcified bone, and report a process that gives reliable, dense labeling after methylmethacrylate (MMA) embedding. Thin sections were processed for postembedding protein Agold immunolabeling with antibodies to rat bone sialoprotein (BSP) and osteopontin (OPN). The density of gold particles over bone was quantified. The density and distribution of immunolabeling for BSP and OPN respectively, were comparable between MMA and LR White.

Reference:

Laboux, O.; Ste-Marie, L.-G.; Glorieux, F. H., and Nanci, A.: Quantitative Immunogold Labeling of Bone Sialoprotein and Osteopontin in Methylmethacrylate-embedded Rat Bone. J. Histochem. Cytochem., 51, 61-7 (2003).

Abstract (courtesy of the Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/51/1/61

Sensitivity is a primary concern in immunohistochemical procedures, but control of background staining is an important secondary factor. Kim and co-workers have investigated methods for minimizing background with tyramide signal amplification in immunohistochemical applications; they find that trypton casein peptone is the most effective blocking agent, and distilled water with Tween-20 the most effective rinsing solution.

Reference:

Kim, S. H.; Shin, Y. K.; Lee, K. M.; Lee, J. S.; Yun, J. H., and Lee, S. M.: An Improved Protocol of Biotinylated Tyramine-based Immunohistochemistry Minimizing Nonspecific Background Staining. J. Histochem. Cytochem., 51, 12932 (2003).

Abstract (courtesy of the Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/51/1/129

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