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Updated: February 12, 2004

N A N O P R O B E S     E - N E W S

Vol. 5, No. 2          February 12, 2004


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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Gold-Labeled Liposomes Reveal HDL Cholesterol Trafficking

A useful protocol for the preparation and use of Nanogold®-labeled liposomes has been reported by Williams and co-workers, who used using DPPE-Nanogold-labeled discoidal HDL liposomes to study receptor-mediated trafficking of cholesterol between lipoproteins and cells. While LDL receptor-mediated endocytosis is relatively well-understood, little is known about the trafficking of high-density lipoprotein (HDL) cholesterol by scavenger receptor BI (SR-BI), an HDL receptor that mediates HDL cholesteryl ester uptake in the selective transfer of HDL lipids to the cell membrane without the uptake and degradation of the HDL particle. Fluorescence and gold labeling was used to localize cell surface sites where the HDL cholesterol trafficking occurs.

Fluorescence confocal microscopy showed SR-BI in patches and small extensions of the cell surface that were distinct from sites of caveolin-1 expression. Electron microscopy was used to localize SR-BI using immunogold labeling with colloidal gold-labeled antibodies, and to localize HDL liposomes using two approaches: immunogold staining of biotinylated liposomes, and direct detection of gold-decorated liposomes prepared using DPPE-Nanogold, enhanced using gold enhancement.

Gold-tagged reconstituted 11 nm discoidal HDL liposomes were prepared using the method of Sparks: palmitoyloleolylphosphatidylcholine (POPC) containing 1 % mol/mol DPPE-Nanogold in trichloromethane was added to a 15-ml conical glass tube and dried under nitrogen. TRIS-saline (0.01 M Tris, 0.15 M NaCl, pH 8) was added to give a 20 mM POPC concentration, and the mixture was vortexed thoroughly. Sodium cholate in TRIS-saline was added (molar ratio POPC/cholate = 0.74) and the mixture vortexed for a further 3 min. The dispersion was then incubated at 37°C and vortexed every 10 min for 1.25 h until completely clear. The desired amount of apoA-I was added (cholate:apoA-1 molar ratios of 34:1, 68:1, 100:1, or 135:1) and the mixture was diluted to 1 mg/mL protein with Tris buffer, then incubated for 1 h at 37°C. Sodium cholate was removed by incubation with hydrated Bio-Beads (1 g of Bio-Beads per 2 mg of cholate) for 2.5 h at 4°C and the Bio-Beads removed by microfiltration through a 0.22-micrometer filter. Protein and lipid recoveries with this method are generally greater than 85%. The reconstituted gold-HDL was purified from unincorporated DPPE-Nanogold by gel filtration over a Superose 6 column (Pharmacia). Stoichiometry of gold incorporation, determined by the UV / visible absorbances at 420 nm (Nanogold) and 280 nm (apoA-I) ranged from 0.5 to 0.8 Nanogold/HDL particle.

For labeling experiments using gold HDL, cells were incubated with gold HDL (10 micrograms/mL) at 4°C for 90 min. Cells were fixed with 2 % glutaraldehyde / 1 % paraformaldehyde in PBS for 30 min and then gold-enhanced according to company protocol. Samples were postfixed with reduced osmium tetroxide (2 % OsO4 / 3 % potassium ferrocyanide) for 90 min, rinsed 3 X 10 min in 0.1 M phosphate buffer, pH 7.4 and 3 X 10 min in deionized water, and en bloc stained with aqueous 1 % uranyl acetate for 1 h. After rinsing 3 X 10 min in deionized water, samples were dehydrated through an ascending series of ethanol and embedded in Durcupan between sheets of Aclar. Ultrathin sections (6090 nm) were picked up on formvar-coated nickel slot grids, and poststained with 1 % methanolic uranyl acetate and 0.3 % aqueous lead citrate.

Both immunolabeling and gold lipid localization were observed in patches or clusters primarily on microvillar extensions of the plasma membrane. These observations suggest that this microvillar domain is a way station for cholesterol trafficking between HDL and cells. SR-BI is not strongly associated with classical membrane rafts rich in detergent-resistant saturated phospholipids, and it is suggested that SR-BI is in a more fluid membrane domain that favors rapid cholesterol flux between the membrane and HDL.

Reference:

Peng, Y.; Akmentin, W.; Connelly, M. A.; Lund-Katz, S.; Phillips, M. C., and Williams, D. L.: Scavenger receptor BI (SR-BI) clustered on microvillar extensions suggests that this plasma membrane domain is a way station for cholesterol trafficking between cells and high-density lipoprotein. Mol. Biol. Cell., 15, 384-396 (2004).

Abstract (courtesy of MBC Online):
http://www.molbiolcell.org/cgi/content/abstract/15/1/384

References for liposome preparation:

More information:

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Nanoprobes at USCAP: Brightfield ISH Staining for HER2 Amplification

In collaboration with Dr. Raymond Tubbs and group at the Cleveland Clinic Foundation, we will be presenting two posters at the upcoming US-Canadian Academy of Pathology Annual Meeting in Vancouver describing further advances in novel staining methods for in situ hybridization. In the first presentation (abstract #205), methods will be described for simultaneous immunohistochemical detection of overexpression of the HER2 oncoprotein and in situ hybridization assessment of HER2 gene using our novel enzyme metallography detection protocol for observation in the conventional brightfield light microscope in a series of 114 invasive ductal breast carcinomas. See poster #18, to be presented on the afternoon of Monday, March 8.

A second poster (abstract #1499) will be presented on Monday morning, March 8. This poster, Stowell-Orbison #204, describes the validation of gold-facilitated in situ hybridization (GOLDFISH), an in situ hybridization method in which detection is achieved with Nanogold®-labeled streptavidin and gold enhancement. HER2 gene amplification in a series of 103 invasive breast carcinomas was quantitatively analyzed using an automated image analyzer, using color and morphometry to quantify signal number within the cell nuclei.

References for GOLDFISH:

More information:

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Silver-Enhanced Undecagold Probes Show Mitochondrial DNA Uptake

Although we recommend our 1.4 nm Nanogold® probes for most electron microscopy applications, we also offer the smaller 0.8 nm undecagold label for applications where a smaller probe size is critical, or where higher resolution methods are available. However, Flierl and co-workers have recently shown that it can also be silver enhanced and used for regular transmission electron microscopy. They undecagold labeling to demonstrate a novel method for importing DNA into mitochondria in living cells, an application for which the small size proved critical.

An N-terminal mitochondrial targeting peptide was conjugated to a short (20-mer) PNA backbone which was then annealed with a complementary oligonucleotide to form a very stable PNA-Peptide/Oligonucleotide (PPO)-Complex, which was shown to be taken up and incorporated into mitochondria in myoblasts. Oligonucleotides labeled with fluorescein or digoxigenin were subsequently localized to mitochondria using fluorescence or immunofluorescence microscopy; undecagold-labeled PPOs were then localized using silver enhancement followed by electron microscopy.

Oligonucleotides were synthesized complementary to the PNA and extended for 3 to 5 nucleotides beyond the PNA sequence at the 5' end. 5' ends of oligonucleotides were modified by C6 S-S phosphoramidite coupled to either FAM (5-carboxyfluorescein), Digoxigenin (DIG) or Monomaleimido-Undecagold. 5' thiolated oligonucleotides labeled with Undecagold were purified by DEAE ion exchange chromatography and analyzed by denaturing TBE-Urea gel electrophoresis. Adherent mouse C2C12 cells were transformed with Undecagold-labeled PPO-complexes for 4 hours in the presence of streptolysin-O or branched polyethylenimine (PEI) with molecular weights of 750 kDa, 25 kDa and 700 Da. Cells were then removed from their substrate, washed and prepared for transmission electron microscopy using the silver enhancement procedure of Humbel.

Reference:

Flierl, A.; Jackson, C.; Cottrell, B.; Murdock, D.; Seibel, P., and Wallace, D. C.: Targeted delivery of DNA to the mitochondrial compartment via import sequence-conjugated peptide nucleic acid. Mol. Ther., 7, 550-557 (2003).

Abstract (Medline):
LINK

Silver enhancement procedure:

Humbel, B. M.; Sibon, O. C.; Stierhof, Y. D.; and Schwarz, H.: Ultra-small gold particles and silver enhancement as a detection system in immunolabeling and in situ hybridization experiments. J. Histochem. Cytochem., 43, 735-737 (1995).

You can also use undecagold for:

  • STEM microscopy.
  • Image analysis.
  • Diffraction studies.
  • Heavy atom derivatization of large proteins or membrane proteins in crystallography.
  • As a high-resolution microscopy size standard.

More information:

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Nanogold® Labeling for MALDI Mass Spectrometry, PAGE and SEM

Horneffer and co-workers investigated the feasibility of using high resolution field emission scanning electron microscopy (FE-SEM) to investigate analyte incorporation into, and distribution within, slowly grown crystals of 2,5-dihydroxybenzoic acid (2,5-DHB) and 2,6-dihydroxybenzoic acid (2,6-DHB). Both are used as a matrix for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). To investigate matrixanalyte interaction in 2,5-DHB crystals, two gold labels (20 nm colloidal gold and Nanogold®) were examined as potential protein markers to visualize proteins in matrix solids by SEM.

2,5-DHB and 2,6-DHB were purified by activated carbon and recrystallized from an aqueous solution. Aliquots of BSA were labeled in house with sulfo-succinimido Nanogold. To achieve a suitable labeling, a Nanogold-protein ratio of 5:1 was chosen. Column gel chromatography with Sephadex G-50 eluted with bi-distilled water was used to separate unbound Nanogold from unlabeled and labeled BSA proteins. Nanogold-labeled BSA was analyzed by MALDI-MS and SDSPAGE. SDS-PAGE confirmed successful labeling. The Nanogold-BSA conjugate was observed in the MALDI mass spectrum, unlike the larger colloidal gold; peaks were also observed corresponding to unlabeled BSA and unconjugated Nanogold, suggesting either incomplete separation after conjugation, or some dissociation under the MALDI conditions used.

2,5-DHB crystals were grown by dissolving 2,5-DHB bi-distilled water in a concentration of 60 g/L; 2,6-DHB was dissolved to saturation concentration of 10 g/L in bi-distilled water; Both matrix solutions were sonicated for 15 minutes for faster dissolution and for release of solvated gas molecules, then placed in a water bath at 36°C for 1 h. Analyte molecules were dissolved in bi-distilled water separately and added to the matrix solution to give a molar analyte-to-matrix ratio of 0.0001 for all protein samples. Solutions were continuously cooled from 36°C down to 4°C without stirring over 1, 24, or 72 h (cooling rates of 32, 1.3, and 0.44 K/h, respectively). Crystals were harvested by filtration, washed with bi-distilled water at 4°C and dried at 30°C for 24 h.

For SEM, Nanogold-BSA doped crystals of 2,5-DHB were cleaved mechanically with a scalpel parallel to their largest surface. The freshly opened inner faces were mounted on aluminum specimen stubs using a small amount of electrically conductive carbon glue, with cleaved surface upwards. After 2 h air-drying, samples were rotary shadowed at room temperature with about 2 or 5 nm Pt/C. Micrographs were recorded at acceleration voltages from 3 up to 8 kV using secondary electron (SE) and back scattered electron (BSE) mode, respectively. For some crystals, the other half was analyzed by X-ray diffraction to investigate the quality of single crystal patterns.

20 nm colloidal gold did not label all the analyte sites in the crystals, due to the presence of unlabeled protein, and therefore the covalently linked and chromatographically purified Nanogold conjugate seemed more promising. However, although Nanogold may be observed by SEM, it was found that the higher magnification required to do so destroyed the 2,5-DHB crystal. Enlargement of Nanogold using gold enhancement may be helpful for SEM applications, since gold gives a much higher backscatter signal than silver; in addition, Nanoprobes is currently developing larger, covalently linked gold labels which may be useful for similar applications. Some preliminary results are linked below.

Reference:

Horneffer, V.; Reichelt, R., and Strupat, K.: Protein incorporation into MALDI-matrix crystals investigated by high resolution field emission scanning electron microscopy. Int. J. Mass Spec., 226, 117-131 (2003).

Abstract (courtesy of Science Direct):
LINK

More information:

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Useful Pre-Embedding Nanogold® Labeling and Gold Toning Protocol

Suikkanen and group report a handy pre-embedding protocol using Nanogold immunolabeling and silver enhancement with gold toning, which confers excellent thermal stability as well as resistance to osmium etching. This was used to study the effects of endocytosis-modulating drugs upon the release of canine parvovirus (CPV) from endosomal vesicles during infection of cultured cells, and the role of phospholipase A2 (PLA2) in this process; electron microscopic localization of the virus after exposure to the different drugs showed that PLA2 activity is essential, and suggested that while endosomes remain intact after infection, it causes changes in permeability which are essential for release.

Norden Laboratories feline kidney cells were incubated with CPV (m.o.i. 50) in Dulbeccos modified Eagles medium for 20 h in the presence or absence of the drugs; when used, they were added on the top of the cells 30 min before virus and maintained until fixation.

The pre-embedding procedure is:

  1. After 120 h, wash dishes with 0.1 M phosphate buffer, pH 7.4. Fix cells with prednisolone phosphate (PLP) for 2 h at room temperature.
  2. After rinsing, permeabilize cells for 8 min at room temperature with phosphate buffer containing 0.01 % saponin and 0.1 % bovine serum albumin, or with 0.05 % Triton X-100 in 0.1 M phosphate (experiments to visualize nuclear antigens).
  3. Incubate primary antibody (MaCPV or RaVP1) on the top of the cells for 1 h at room temperature.
  4. Wash with permeabilization buffer.
  5. Incubate with Nanogold-labeled IgG (anti-rabbit or anti-mouse) secondary antibody on the top of the cells for 1 h at room temperature.
  6. Wash with permeabilization buffer.
  7. Postfix with 1 % glutaraldehyde in 0.1 M phosphate buffer for 10 min at room temperature. Quench with 50 mM ammonium chloride in phosphate buffer, and wash with both phosphate buffer and water.
  8. Enhance in the dark with HQ Silver for 2 min, then wash thoroughly with water.
  9. 2 % sodium acetate, 3 X 5 min.
  10. 0.05 % gold chloride 10 min on ice.
  11. 0.3% sodium thiosulphate 2 X 10 min on ice.
  12. Wash with water.
  13. Postfix with 1% osmium tetroxide in 0.1 M phosphate buffer for 1 h at 4°C.
  14. Dehydrate with a descending concentration series of ethanol.
  15. Stained with 2 % uranyl acetate.
  16. Embed by placing plastic capsules filled with Epon LX-112 upside-down on top of the cells. After polymerization, warm up to 100°C, carefully remove capsules, and cut 50 nm horizontal sections.
  17. Stain with 2% uranyl acetate and lead citrate.

Reference:

Suikkanen, S.; Antila, M.; Jaatinen, A.; Vihinen-Ranta, M., and Vuento, M.: Release of canine parvovirus from endocytic vesicles. Virology, 316, 267-280 (2003).

Abstract (Medline):
LINK

More information:

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Other Recent Publications

Furuya and co-workers report a protocol for staining a single slide with up to 50 antibodies at once using a novel device called a "multiplex-immunostain chip (MI chip)." The chip is a panel of antibodies contained in a silicon rubber plate with 50 2-mm-diameter wells. A tissue section slide is placed on the plate and is fastened tightly with a specially designed clamp. The plate with the slide is then turned upside down, which applies the antibodies to the section. This technology allows staining with 50 different antibodies simultaneously, reducing the time, effort, and expense of immunohistochemical analysis.

Reference:

Furuya, T.; Ikemoto, K.; Kawauchi, S.; Oga, A.; Tsunoda, S.; Hirano, T., and Sasaki, K.: A novel technology allowing immunohistochemical staining of a tissue section with 50 different antibodies in a single experiment. J. Histochem. Cytochem., 52, 205-210 (2004).

Abstract (courtesy of the Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/52/2/205

In the same issue, LpezCepero reports a sensitive procedure for staining mitochondria using silver carbonate followed by gold toning and sodium thiosulfate fixation, which provides a high level of heterogeneity among mitochondria. This is thought to reflect different stages in their life cycle, and might be used to discriminate older mitochondria.

Reference:

LpezCepero, J. M.: Silver Carbonate Staining Reveals Mitochondrial Heterogeneity. J. Histochem. Cytochem., 52, 211-216 (2004).

Abstract (courtesy of the Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/52/2/211

Liu and group compare pre-embedding labeling with Nanogold and post-embedding labeling with 10 nm colloidal gold to explore Nogo-A reactivity at rat synapses. Immunogold particles for Nogo-A could be identified in dendrites of the spinal cord motoneurons, with some in close apposition to the postsynaptic density and the postsynaptic dense bodies at both symmetric and asymmetric synapses. These findings provide a morphological basis for the idea that the Nogo-ANogo receptor can contribute to structural plasticity at synapses as well as along the axonal pathway.

Reference:

Liu, Y.-Y.; Jin, W.-L, Liu, H.-L., and Ju, G.: Electron microscopic localization of Nogo-A at the postsynaptic active zone of the rat. Neurosci. Lett., 346, 153-156. (2003).

Abstract (Medline):
LINK

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