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Updated: July 11, 2003

N A N O P R O B E S     E - N E W S

Vol. 4, No. 7          July 11, 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® Helps Demonstrate Hierarchical Folding in Chromosomes

A combined fluorescence and immunogold study using Nanogold®-Fab' fragments to immunolabel components of chromosomes has demonstrated the presence of large-scale hierarchical folding in metaphase chromosomes containing altered scaffold-associated region (SAR) sequence composition. Chinese hamster ovary cells were isolated containing high density insertions of a transgene containing lac operator repeats and a dihydrofolate reductase gene, with or without flanking SAR sequences. Lac repressor staining provided high resolution labeling with good preservation of chromosome ultrastructure, and the study demonstrates how the unique resolution and penetration of Nanogold conjugates enable labeling nuclear features in cells and tissues.

Transfected cells were fixed without permeabilization for 24 h in 1.6% freshly prepared paraformaldehyde in calcium and magnesium-free PBS. To increase staining, long incubation times (21 h) at 4°C in a wet chamber were used for both primary anti-lac repressor and secondary Nanogold-Fab' antibodies. Freshly cut Epon sections were silver enhanced, dehydrated, and embedded, and stained with lead citrate and uranyl acetate. Thick sections (0.4 mm) were examined using a transmission electron microscope (TEM). Fluorescence labeling showed that insertions did not change chromatid diameter; ultrastructural analysis of electron microscopy data demonstrated that the region stained by anti-lac repressor antibodies assumes a regular size with sharply defined parallel faces, supporting its large-scale organization into a folding subunit about 250 nm in diameter. metaphase chromosomes. This contradicts predictions of simple radial loop models, and provides the first unambiguous demonstration of a hierarchical folding subunit above the level of the 30-nm fiber within normally condensed metaphase chromosomes.

Reference:

Strukov, Y. G.; Wang, Y., and Belmont, A. S.: Engineered chromosome regions with altered sequence composition demonstrate hierarchical large-scale folding within metaphase chromosomes. J. Cell Biol., 162, 23-35 (2003).

Abstract (courtesy of the Journal of Cell Biology): http://www.jcb.org/cgi/content/abstract/162/1/23

The electron microscopy immunolabeling method is described in more detail in earlier publications from this group:

More information:

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Nanovan and Nano-W: 'Tunable' Negative Staining

Unlike 'positive' stains, which stain regions of specimens, negative stains fill in the gaps between features and contrast the edges of particulate or suspended specimens such as protein complexes. Nanoprobes offers two negative stain reagents with complementary properties. NanoVan is recommended for use with Nanogold® because it is based on vanadium and is therefore less electron-dense than heavy metal based stains such as uranyl acetate or lead citrate. Nano-W is based on the heavier element tungsten and therefore gives a more dense stain, and is more suited to use with larger gold labels. These two reagents are completely miscible, and therefore may be mixed together in different proportions to generate any desired intermediate stain density.

Both have near-neutral pH, and NanoVan has been found to be less susceptible to electron beam damage than uranyl acetate. Franzetti and colleagues found NanoVan's pH of close to 8 was better suited to their system than that of uranyl acetate, and used negative stain electron microscopy and image analysis of 1,300 particles to define the structure of a large, tetrahedral, dodecameric protease complex (TET) from archaea. TET, which has broad aminopeptidase activity and can process peptides of up to 30-35 amino acids in length, was found to have a central cavity accessible through four narrow channels (<17 Å wide) and four wider channels (21 Å wide).

Reference:

Franzetti, B.; Schoehn, G.; Hernandez, J. F.; Jaquinod, M.; Ruigrok, R. W.; and Zaccai, G.: Tetrahedral aminopeptidase: a novel large protease complex from archaea. EMBO J., 21, 2132-2138 (2002).

Abstract (courtesy of the EMBO Journal):
http://emboj.oupjournals.org/cgi/content/abstract/21/9/2132

Jeon and Ishikawa used Nano-W to determine the structure of thioredoxin peroxidase (ApTPx) of the aerobic hyperthermophilic archaeon Aeropyrum pernix. ApTPx was prepared by cloning a gene (APE2278) encoding the peroxiredoxin (Prx) homologous protein of yeast and human to produced the encoded protein in E. coli cells. Purified ApTPx (wild type and experimental mutants lacking significant cysteine residues) were diluted with 50 mM Tris-HCl, pH 7.5, to final protein concentrations of 50 micrograms/ml. 10 microliters was applied onto a glow-discharged carbon-coated grid, the samples negatively stained with Nano-W for 1 min, and then excess liquid was removed with blotting paper and the grids were examined with a Hitachi H-9000 transmission electron microscope operating at 100 kV. Analysis of the EM and HPLC data showed ApTPx to be a hexadecameric protein forming 2-fold toroid-shaped structure with outer and inner diameters of 14 and 6 nanometers, respectively. This indicated that ApTPx is a novel hexadecameric protein composed of two identical octamers.

Jeon, S. J., and Ishikawa, K.: Characterization of novel hexadecameric thioredoxin peroxidase from Aeropyrum pernix K1. J. Biol. Chem., 278, 24174-24780 (2003).

Abstract (courtesy of the Journal of Biological Chemistry):
http://www.jbc.org/cgi/content/abstract/278/26/24174

Fry and co-workers have also used Nano-W, with 6 nm protein-A gold in the molecular characterization of a novel human centrosomal protein, C-Nap1 (for centrosomal Nek2-associated protein 1). Centrosomes diluted 1:10 in PBS were sedimented onto Pioloform and carbon-coated grids blocked with 0.1% gelatin in PBS using an airfuge (65,000 g-av, 15 min), incubated with affinity-purified antiC-Nap1 IgGs (R63, 1 microgram/ml, 45 min) followed by protein A6 nm gold, then negatively stained with Nano-W.; immunoelectron microscopy revealed that C-Nap1 is associated specifically with the proximal ends of both mother and daughter centrioles. C-Nap1 was concentrated at centrosomes in all interphase cells, but immunoreactivity at mitotic spindle poles was strongly diminished, supporting a proposed model implicating both Nek2 and C-Nap1 in the regulation of centriolecentriole cohesion during the cell cycle.

Reference:

Fry, A. M.; Mayor, T.; Meraldi, P.; Stierhof, Y.-D.; Tanaka, K., and Nigg, E. A.: C-Nap1, a novel centrosomal coiled-coil protein and candidate substrate of the cell cycle-regulated protein kinase Nek2. J. Cell Biol., 141, 1563-74 (1998).

More information:

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Nanogold® and Colloidal Gold, Pre- and Post-Embedding Immuno-EM

Liu and co-workers provide a useful comparison of pre-embedding and post-embedding gold labeling in their recent paper on the immunoreactivity of the membrane-associated protein Nogo-A at rat spine synapses. Nogo, a member of the reticulon family of membrane-associated molecules, has three isoforms, Nogo-A, -B, and -C. Nogo-A is a prominent myelin-derived inhibitor of axonal outgrowth, and monoclonal antibody IN-1 raised against Nogo-A has successfully induced fiber regeneration and sprouting with functional recovery in injured spinal cord as well as in ischemic cortex. Upregulating the transgenic expression of Nogo-A in peripheral nerves obstructs axonal regeneration and functional recovery after sciatic nerve crush, implying that Nogo-A may override growth-permissive and -promoting effects in the lesioned peripheral nerve.

Nanogold was used for pre-embedding labeling in 50 micron rat spinal cord sections. After cryoprotection for 1 hour in 0.01 M phosphate buffered saline (PBS, pH 7.4) containing 25% sucrose and 10% (v/v) glycerol and freezethaw treatment, they were blocked with 0.01 M PBS containing 5% bovine serum albumin and 5% normal goat serum for 4 h. The sections were then stained with rabbit anti-Nogo-A antibody generated against a Nogo-A specific C-terminus 572-amino acid fragment (6211192), diluted to 1:300 in 1% bovine serum albumin and 1% normal goat serum for 48 h at 4°C. After washing in 0.01 M PBS, they were incubated with Nanogold-IgG goat anti-rabbit IgG diluted in 1:100 at room temperature overnight. After rinsing and postfixation with 2% glutaraldehyde in 0.01 M PBS for 45 min, silver enhancement was performed using HQ Silver. The sections were fixed with 0.5% osmium tetroxide in 0.1 M phosphate buffer for 1 h, dehydrated with graded ethanol, replaced with propylene oxide, and flat-embedded in Epon 812.

For post-embedding labeling, 10 nm gold was used in 100 micron tissue sections. These were trimmed into small pieces, cryoprotected, then rapidly frozen in liquid propane, transferred to pre-cooled anhydrous methanol containing 0.5% uranyl acetate in a cryosubstitution unit, and after the temperature was set to rise stepwise to -45°C, infiltrated with Lowicryl HM 20 resin and polymerized under ultraviolet light for 48 h. Ultrathin sections were mounted on nickel grids, treated with a saturated NaOH solution in absolute ethanol for 23 s, rinsed and incubated in 0.1% sodium borohydride and 50 mM glycine in Tris with 0.05M NaCl and 0.1% Triton X-100 (TBNT) for 10 min, and blocked with 2% bovine serum albumin in TBNT for 20 min. The sections were then incubated overnight in rabbit anti-Nogo-A antibody, diluted 1:300 in 2% bovine serum albumin in TBNT overnight at 4°C. After rinsing, the sections were placed in 2% bovine serum albumin for 20 min and then 10 nm gold goat anti-rabbit IgG, diluted to 1:40 in TBNT containing 2% bovine serum albumin and 0.5 mg/ml polyethyleneglycol. After air-drying, the sections were counterstained with uranyl acetate and lead citrate.

Pre-embedding immunogold-silver cytochemistry with Nanogold revealed more antigen immunoreactivity than post-embedding immunogold cytochemistry with 10 nm gold, supporting the enhanced ability of Nanogold conjugates to penetrate into tissues; however, post-embedding labeling revealed more accurate antigen localization, suggesting that this technique better preserves ultrastructural morphology. However, Nanogold has also been successfully used for post-embedding labeling, and may combine increased labeling density with better penetration in this application.

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):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12853107&dopt=Abstract

More information:

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Nanoprobes at Microscopy & Microanalysis '03

Some preliminary results using a novel combined fluorescent and gold-conjugated neuronal tracer will be presented at Microscopy & Microanalysis '03 in San Antonio, Texas, August 3 - 7. A paper entitled "Colloidal Gold Conjugate of Recombinant Cholera Toxin B-Subunit of Alexa Fluor and Dextran-Texas Red-Nanogold Fluorescent Dyes for Use in Correlative Microscopy and Intravital Imaging," will be presented by Dr. Eduardo Rosa-Molinar of the University of Puerto Rico-Rio Piedras at 3:00 pm on Wednesday, August 6 in Room 201, as part of a symposium on "Advances in Bioimaging."

This application was briefly discussed before (see our 1998 MSA paper below); the 1.4 nm Nanogold® particle and Texas red were both cross-linked to an amino-functionalized dextran to give a non-targeted tracer which was then localized using both fluorescence and electron microscopy.

More information:

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Other Recent Publications

FolletGueye and co-workers have reported an improved fixation method for the immunogold localization of green fluorescent protein (GFP). The method is a simple one-step procedure which consists of fixing in the dark for 30 min, 60 min, or 120 min at 4C in a solution consisting of 1% glutaraldehyde and 1% osmium tetroxide in 0.1 M Na cacodylate buffer, pH 7.2. This mixture was prepared and used immediately. After washing in distilled water, the samples were gradually dehydrated in 10%, 20%, and 40% aqueous ethanol (10 min in each bath), then in 60% and 80% (20 min in each bath), and finally in anhydrous ethanol for 30 min, and embedded in LR White or Spurr resin. GFP-tagged Golgi glycosyltransferase was localized in transgenic BY-2 cells using rabbit anti-GFP primary and 10 nm gold anti-rabbit secondary antibodies; this method allowed improved structural preservation of the endomembrane system, and anti-GFP antibodies bound with high specificity, allowing the localization of GFP-tagged glycosyltransferases within individual Golgi cisternae.

Reference:

Follet-Gueye, M. L.; Pagny, S.; Faye, L.; Gomord, V., and Driouich, A.: An improved chemical fixation method suitable for immunogold localization of green fluorescent protein in the Golgi apparatus of tobacco Bright Yellow (BY-2) cells. J. Histochem. Cytochem., 51, 931-940 (2003).

Abstract (courtesy of the Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/51/7/931

In the same issue, Umland and group report an optimized HOPE (HEPESglutamic acid buffer-mediated organic solvent protection effect) fixation protocol, previously used for ISH targeting mRNA in lung tissues, applied for ISH and immunocytochemistry on cultured cells. HOPE can be used with cells and tissues and with a broad spectrum of immuno-histocytochemical and molecular techniques.

Reference:

Umland, O.; Ulmer, A. J.; Vollmer, E., and Goldmann, T.: HOPE fixation of cytospin preparations of human cells for in situ hybridization and immunocytochemistry. J. Histochem. Cytochem., 51, 977-980 (2003).

Abstract (courtesy of the Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/51/7/977

As part of their studies to devise a strategy using human autoimmune phage display libraries to generate reagents for biological studies of conserved cellular proteins, Farneas and Ditzel have prepared their own combined fluorescent and gold-labeled Fab fragments of recombinant antibodies against Annexin XI. This protein, a member of the Ca2+-dependent phospholipid binding protein family, was recently identified as an autoantigen targeted by autoantibodies in several systemic autoimmune diseases. The novel probe was prepared by sequential labeling with fluorescein isothiocyanate (FITC) followed by Nanogold®, and used to help elucidate the cellular role of Annexin IX by confocal and electron microscopy.

Reference:

Farneas L., and Ditzel, H. J.: Dissecting the cellular functions of annexin XI using recombinant human annexin XI-specific autoantibodies cloned by phage display. J. Biol. Chem., e-publication ahead of print, manuscript M210852200v1 (2003).

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