N A N O P R O B E S     E - N E W S

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).

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:

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).

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).

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).

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).