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N A N O P R O B E S     E - N E W S

Vol. 10, No. 6          June 29, 2009


Updated: June 29, 2009

In this Issue:

This monthly newsletter is to inform you about techniques to improve your immunogold labeling, highlight interesting articles and novel applications of metal nanoparticles, and answer your questions. We hope you enjoy it and find it useful, as always, let us know if we can improve anything.

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Double Labeling with Sequential Nanogold® and Gold Enhancement

Multiple labeling is one of the major challenges in immunogold technology, and typically requires the use of colloidal gold particles of two different, distinct sizes. However, as previously reported, double labeling can also be achieved by the use of our 1.4 nm Nanogold immunogold conjugates, combined with differential gold enhancement: two Nanogold immunolabeling reactions, used with sequential gold enhancement steps, one after labeling of the first target with Nanogold and the second after labeling the second target, can be used to give two populations of gold particles with non-overlapping sizes.
Gold enhancement is an alternative to silver enhancement, developed by Nanoprobes. In gold enhancement, gold nanoparticles, colloidal gold or gold cluster labels are enlarged by the catalytic deposition of gold from solution. The process is similar to silver enhancement, but has important advantages for many applications:

  • Safe for use before any strength osmium tetroxide - gold enhanced particles are not etched, as silver-enhanced gold can be (especially with uranyl acetate).
  • Compatible with physiological buffers: does not precipitate with halides, as silver can do.
  • More selective reaction: compatible with metal substrates for cell culture or biomaterials.
  • Less pH sensitive than silver enhancement: can be used in a wider pH range.
  • Gold gives a much stronger backscatter signal than silver: better for SEM labeling.
  • Near neutral pH for best ultrastructural preservation.
  • Low viscosity, so the components may be dispensed and mixed easily and accurately.
  • Cleaner signals with lower background for many light microscopy and blotting applications.

[Gold enhancement schematic, and double labeling by sequential Nanogold immunolabeling and gold enhancement (119k)]

(left) The gold enhancement reaction, showing the progressive enlargement of Nanogold. (right) Scheme for double labeling using differential gold enhancement of Nanogold: (a) immunolabeling of first target using Nanogold-antibody; (b) first gold enhancement reaction (12 minutes); (c) immunolabeling of second target with Nanogold probe; (d) second gold enhancement reaction (4 minutes) gives two distinct, non-overlapping populations of gold particles of different size.

In their recent paper in Cerebral Cortex, Paspalas and co-workers expand on their double labeling method, which they used to determine the cellular and subcellular expression of Major Vault Protein (MVP) in primate and rodent cerebral cortex, and in cortical neurons in vitro. MVP is a 100-kDa protein which complexes with small-untranslated vault RNA(vRNA), telomerase-associated protein and poly(ADP-ribose) polymerase, in structures known as vault particles. These are the largest known ribonucleoprotein particle, about three times the size of ribosomes, weighing 13 MDa and measuring approximately 65 x 40 nm. Vault particles are so named because of their morphology, comprising two identical hemispherical halves, each consisting of a central ring enclosed by a radially symmetric 8-petaled flower. MVP is highly conserved in eukaryotic cells and upregulated in a variety of tumors, correlating with poor prognosis. Although vault proteins have been speculated to function as cargo transporters in several cell lines, no work to date has characterized the protein in neurons. The authors studied MVP organization and function in normal, drug-naive neurons using a combination of immunogold high-resolution electron microscopy in monkey and rat cerebral cortex with immunocytochemical and molecular analyses of primary cortical cultures.

A variety of different light and electron microscopic methods were used to localize MVP, utilizing Nanogold labeling with both silver and gold enhancement. For electron microscopy, sections were placed in anti-MVP for 48 hours, then incubated with biotinylated F(ab')2, both diluted in tris-buffered aline (TBS) with 2% normal goat serum (NGS), 0.1% acetylated BSA-C (Aurion), and 0.01% Tween 20. Next, the biotinylated antibody was detected for 2 hours in 1:200 Nanogold-labeled goat anti-biotin antibody. The two-layer immunocomplex was then fixed for 5 minutes in 1% phosphate-buffered glutaraldehyde and, after a thorough wash in ultrapure water, enhanced for 810 minutes on ice with a HQ Silver. This procedure produces multiparticle aggregates; therefore, an alternative approach was also used: to directly complex MVP antibody with nanogold-Fab', followed by silver enhancement. Because the 2-layer procedure has a stoichiometry close to 1 : 1 (i.e., one particle per IgG fragment), it is expected to yield single immunoparticles.

The double labeling procedure was used to control for specificity. First, sections were preincubated for 30 minutes in N-TBS supplemented with 0.1% acetylated BSA (Aurion), 0.1% fish skin gelatin and 0.07% Tween-20 ("gold buffer"), and anti-MVP was complexed with Nanogold-Fab' (diluted 1:200 in gold buffer). After washing in ultrapure water and 20 mM sodium citrate, the Nanogold was enhanced for 12 minutes on ice with GoldEnhance EM. Subsequently, a second incubation with Nanogold-Fab' (1:200 dilution) was conducted for 4 hours. Sections were finally postfixed in glutaraldehyde and transferred for 4 minutes to a fresh portion of GoldEnhance. This sequential enhancement produced distinct, non-overlapping particle-size groups.

Omission of the bridging biotinylated antibodies abolished immunoreactivity. Similarly, peroxidase labeling was eliminated when blocking the biotinylated probes with avidin/biotin (Vector). To control for self-nucleation of the metallographic developer, Nanogold was omitted, whereas the sections were routinely processed for silver or gold autometallography for 15 minutes at room temperature. In labeling MVP, enhancement never exceeded 12 minutes at 4°C. For electron microscopy, sections were postfixed in 1% buffered osmium tetroxide (15 and 30 minutes for silver- and gold-enhanced material, respectively), treated with ethanolic uranyl acetate en block, and finally embedded in Durcupan epoxy resin (Fluka, Steinheim, Switzerland) and polymerized at 58°C for 48 hours under vacuum. Layers II-IV and V-VI of PFC, and layers II-VI of S1 as well as all strata of CA1 were sampled for re-sectioning and analysis under a JEM1010 (Jeol) transmission electron microscope at 80-100 kV. MVP structures were digitally captured at 45,000-350,000 x magnification (Gatan), and individual panels were adjusted for brightness and contrast.

Immunogold labeled free and attached ribosomes, and structures with a similar appearance to vaults located on the rough endoplasmic reticulum and the nuclear envelope. The nucleus was immunoreactive in association with nucleopores. Axons and particularly principal dendrites expressed MVP along individual microtubules, and in pre- and postsynaptic structures. Synapses were not labeled. Colocalization with microtubule-associated protein-2, tubulin, tau, and phalloidin was observed in neurites and growth cones in culture. Immunoprecipitation coupled with reverse transcription PCR showed that MVP associates with mRNAs that are known to be translated in response to synaptic activity. Taken together, these results provide the first characterization of neuronal MVP along the nucleus-neurite axis, and may offer new insights into its function in the brain.

Reference:

  • Paspalas, C. D.; Perley, C. C.; Venkitaramani, D. V.; Goebel-Goody, S. M.; Zhang, Y.; Kurup, P.; Mattis, J. H., and Lombroso, P. J. Cereb. Cortex., 19, 1666-1677 (2009).

Original reference for Nanogold-GoldEnhance double labeling method:

Original reference for Nanogold-HQ Silver enhancement method:

More information:

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Monofunctionality: does it Always Work?

Our Nanogold® labeling reagents are separated by degree of functionality during their manufacture. Nanogold is prepared using a mixture of non-functional and amino-functionalized organic ligands: this yields a statistical mixture in which some gold particles have no amino groups, some have one, and some have more than one. This mixture is then separated by ion exchange chromatography: the mixture is passed over a negatively ionized gel column, and eluted with buffer of increasing ionic strength, sequentially eluting species with greater numbers of amines.

To prepare Nanogold labeling reagents, we collect the the species eluted using an ionic strength which we have consistently found to elute the second Nanogold peak in the chromatogram (i.e. the first Nanogold species to be eluted after the initial non-binding species has cleared). This behaves chromatographically as a monofunctional species, and when converted to a reactive species such as a maleimido or sulfo-N-hydroxysuccinimido Nanogold derivative, reacts in a 1 : 1 stoichiometry with a variety of biological molecules, under a variety of conditions when mixed in a range of stoichiometric ratios. The "mono" designation is based on its reactivity and behavior, and the fact that it has been separated according to degree of functionalization.

We are sometimes asked whether we can provide greater control over functionality, or provide "strictly monofunctional" reagents. While it may be possible to use more stringent analytical ion exchange protocols, there are no readily available methods to prove that the compounds produced contain only a single reactive group. Chromophoric or fluorescent labels are affected by the presence of the much larger Nanogold particle, which either overlaps with their spectra, or changes their values through energy transfer or other modes of interaction, while enzymatic labels may also be affected by the gold in unpredictable ways. However, we find that in all our test systems, they provide 1 : 1 labeling under most conditions.

If you are experiencing problems isolating 1 : 1 conjugates, particularly if you are labeling small molecules such as peptides, the following suggestions will help you to obtain monofunctional products:

  • Use an excess of the Nanogold reagent over the molecule to be labeled. This will favor the reaction of only one molecule with the Nanogold reagent.

  • Add the molecule to be labeled to the Nanogold: this will ensure that there is always an excess of Nanogold over the molecule to be labeled through the reaction. If you add the Nanogold reagent to the molecule to be labeled, at the start of the addition there will be an excess of the molecule to be labeled over Nanogold: if the reaction is sufficiently fast and any Nanogold species containing more than one reactive group are present, this may lead to multiply conjugated Nanogold species.

  • Consider the possibility that other reaction mechanisms may contribute to multiple labeling, particularly when using Monomaleimido Nanogold. Thiols have a very high affinity for gold, and can displace the ligands used in Nanogold, providing a secondary means of conjugation which can lead to multiple labeling even with strictly monofunctional Nanogold reagents. We have found that conjugation of Fab' antibody fragments occurs both to Nanogold and to larger gold particles stabilized with modified alkylthiols.

    If you have followed the suggestions above and still observe multiple labeling, we recommend switching the conjugation reaction, introducing aprimary aliphatic amine in place of your thiol, and using Mono-Sulfo-N-hydroxysuccinimido Nanogold for the labeling reaction: this will remove the possibility of direct thiol coordination.

  • If you are conducting an amine labeling but are concerned that thiols elsewhere in the molecule (such as cysteine residues) might contribute to multiple labeling, block these using N-ethylmaleimide before Nanogold reaction.

These considerations may also be important in aggregation or precipitation problems: if you are experiencing precipitation or aggregation, particularly during a Monomaleiido Nanogold labeling reaction, consider the possibility that thiol coordination may be a factor.

We will be glad to advise on specific problems: call us at 1-877-447-6266 (US and Canada) or +1-(631) 205-9490, or e-mail us.

More information:

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HIV Nef Protein Targeting Revealed by Nanogold®

Nanogold®-Fab' is the smallest commercially available immunogold probe. It provides the most precise immunolabeling of any commercially availabe probe, with several important advantages:

  • High specimen penetration.
  • Highest resolution.
  • High labeling density.
  • High level of access even to restricted or hindered antigens.

These advantages are maximized when Nanogold probes are combined with the high contrast obtained using gold enhancement, and this was demonstrated again recently by daSilva and group in their paper in the Journal of Virology, which used the method to investigate the role of human immunodeficiency virus type 1 Nef protein.

[Nanogold-Fab' vs. colloidal gold, and Nanogold TEM immunolabeling with gold enhancement (93k)]

(left) Size comparison of Nanogold-Fab' with conventional 5 nm colloidal gold-IgG probe, showing overall probe size and distance of gold from target. (right) Pre-embedding immunolabeling using Nanogold-Fab' and GoldEnhance EM, showing uniform enlarged particles (see: Marra, P.; Salvatore, L.; Mironov, A Jr.; Di Campli, A.; Di Tullio, G.; Trucco, A.; Beznoussenko, G.; Mironov, A., and De Matteis, M. A.; Mol. Biol Cell., 18, 1595-1608 (2007)).

The most highly transcribed viral gene during the early phase of human immunodeficiency virus type 1 (HIV-1) infection does not encode an enzyme or structural protein, but an accessory protein named Nef. The Nef protein of HIV-1 downregulates the CD4 coreceptor from the surface of host cells by accelerating the rate of CD4 endocytosis through a clathrin/AP-2 pathway. However, unlike typical endocytic recycling receptors like the transferrin receptor or the low-density lipoprotein receptor, CD4 that is forcibly internalized by Nef does not return to the cell surface, but is delivered to lysosomes for degradation, and expression of Nef therefore decreases both the surface and total levels of CD4. The authors hypothesized that internalized CD4 is kept from returning to the plasma membrane because Nef could also act on endosomes to direct CD4 to lysosomes.

Immunoelectron microscopy allowed the localization of CD4 at the ultrastructural level, and combined with . The cells were fixed with 4% (wt/vol) PFA in phosphate-buffered saline (PBS) for 1 hour, rinsed with 50 mM glycine in PBS for 15 minutes, then blocked with 1% (wt/vol) bovine serum albumin in PBS. Cells were then permeabilized with 0.05% (wt/vol) saponin and 1% (wt/vol) bovine serum albumin in PBS, incubated with anti-CD4 antibody for 1 hour at room temperature or overnight at 4°C, rinsed, then incubated with Nanogold-labeled secondary antibodies for 1 hour at room temperature. The cells were fixed with glutaraldehyde (2.5% vol/vol in 0.1 M cacodylate buffer) for 1 hour, rinsed and enhanced with freshly mixed GoldEnhance EM for 6 minutes, then postfixed in reduced osmium prior to embedding in Epon. Sections of 70 to 100 nm were stained with lead citrate prior to imaging with a Tecnai 20 transmission electron microscope (FEI) operating at 120 kV. Images were captured on a 2,000- by 2,000-pixel CCD camera (Gatan).

In combination with immunofluorescence and binding studies, the authors found that Nef also acts to promote ubiquitination of CD4, thus tagging it for recognition and collection by the endosomal sorting complex required for transport (ESCRT) machinery which then targets it to the multivesicular body (MVB) pathway for eventual delivery to lysosomes. Perturbation of this machinery was found not to prevent removal of CD4 from the cell surface, but did preclude its lysosomal degradation, and this indicates that accelerated endocytosis and targeting to the MVB pathway are separate functions of Nef. Both CD4 and Nef were found to be ubiquitinated on lysine residues, but this modification is dispensable for Nef-induced targeting of CD4 to the MVB pathway. This provides a significant insight into the mechanism of infection by HIV-1 which may yield new therapeutic targets.

Reference:

  • daSilva, L. L.; Sougrat, R.; Burgos, P. V.; Janvier, K.; Mattera, R., and Bonifacino, J. S.: Human immunodeficiency virus type 1 Nef protein targets CD4 to the multivesicular body pathway. J. Virol., 83, 6578-6590 (2009).

More information:

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EnzMetTM: Best for ISH and IHC

If you have used Nanogold-streptavidin for in situ hybridization (ISH), it's worth taking a look at our new detection technology, EnzMetTM (Enzyme Metalllography). This provides the most sensitive and specific detection both for ISH and for immunohistochemistry (IHC).

Nanoprobes has developed a number of detection technologies for Chromogenic in situ hybridization (CISH) that provide higher sensitivity, lower copy number detection and clearer signals than enzyme chromogens such as DAB, including Nanogold® with silver acetate autometallography and Nanogold with gold enhancement (GOLDFISH). However, EnzMetTM provides both higher sensitivity, virtually zero background, and a level of resolution that allows you to count individual gene copies - all major advantages over previous methods. Advantages include:

  • EnzMetTM technology uses HRP to deposit metallic silver with extraordinary selectivity. Background is virtually zero.
  • High sensitivity: detect single copies of target genes, or low-abundance proteins with almost no background.
  • Virtually no diffusion of reaction product means super-sharp signals with highest resolution. Count individual gene copies.
  • Black, sharply defined, non-diffusing stain lets you clearly see underlying morphology. EnzMetTM is readily distinguished from all counterstains.
  • Does not fade or bleach: can be archived indefinitely.

For clinical applications, EnzMetTM has been licensed to Ventana Medical Systems (now part of Roche) for use as a detection system in automated slide stainers; it is marketed and sold as SISH (Silver In Situ Hybridization). It is already available in Europe and other parts of the world, and is currently awaiting FDA approval in the USA. However, if you are doing in situ hybridization without an automated slide staining platform, you can purchase EnzMetTM reagents from us for research use.

For in situ hybridization, use EnzMetTM HRP Detection Kit for IHC/ISH (catalog number 6001) from Nanoprobes. Complete product information, instructions and protocols for this reagent are available on our web site.

Reference:

  • 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).

A comparison of the results obtained using the different Nanogold and metallographic in situ hybridization methods and using the conventional organic chromogen, diaminobenzidine (DAB) is shown below: the evolution of metallographic chromogenic in situ hybridization, and the improvements provided by EnzMetTM are clearly visible.

[Metallographic in situ hybridization results (93k)]

(a) and (b): Single copies of HPV-16 in SiHa cells detected by tyramide signal amplification (TSA, also known as CARD, or catalyzed reporter deposition) followed by detection with (a) streptavidin-peroxidase developed with DAB, and (b) streptavidin-Nanogold with silver acetate autometallography. Copies of HPV-16 appear as single spots. (H & E counterstain. Original magnification X 560). (c) Nanogold with gold enhancement (GOLDFISH) procedure in tissue with HER2 gene amplification, showing large, confluent nuclear signals from multiple gene copies in close proximity. Nuclear fast red counterstain (original magnification 400). (d): EnzMetTM 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).

EnzMetTM is also highly effective for immunohistochemistry (IHC), and it can be used for highly sensitive detection with minimal background and very high contrast on blots. Immunohistochemical application was tested using high-complexity tissue microarrays (TMAs). 88 common solid tumors were evaluated by enzyme metallography (EnzMet) using an automated slide staining system (Ventana Medical Systems); targets were chosen to assess the ability of EnzMet to specifically localize encoded antigens in the nucleus (estrogen receptor), cytoplasm (cytokeratins), and cytoplasmic membrane (HER2) in TMAs. The intensity of staining for all three antigens evaluated was comparable for breast tumors as well as carcinomas of kidney, colon, and prostate. However, the quality of staining in EnzMet IHC preparations was much sharper, and the stain deposits were better defined, showing a more punctate appearance, than that found with DAB. Full concordance was found between the EnzMet and conventional IHC results. The EnzMet reaction product was dense and sharply defined, did not appreciably diffuse, and provided excellent high-resolution differentiation of cellular compartments in paraffin sections for the nuclear, cytoplasmic, and cell membrane-localized antigens evaluated. The higher density of elemental silver deposited during enzyme metallography permitted evaluation of core immunophenotypes at a relatively low magnification, without the need for oil immersion, allowing more tissue to be screened in an efficient manner. In addition, the signal is stable and provides a permanent record.

Reference:

  • 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).

    For immunohistochemistry, use EnzMet EnzMet HRP Detection Kit for IHC/ISH (catalog number 6001) from Nanoprobes. Complete product information, instructions and protocols for this reagent are available online.

    [EnzMet for IHC (83k)]

    Demonstration of immunohistochemical staining using EnzMetTM. (Left) EnzMet staining of epithelial cytokeratins in paraffin-embedded human prostate adenocarcinoma using a secondary immunoperoxidase method (original magnification x 400). (center) Imunoperoxidase staining of epithelial cytokeratins in paraffin-embedded human bladder tumor: secondary immunoperoxidase with DAB; (right) secondary immunoperoxidase method using EnzMet (original magnification x 400).

    In most cases, the EnzMetTM protocol using EnzMetTM 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 EnzMetTM Detect A. Incubate for 4 minutes.
      Note: Excess water can lead to the dilution of EnzMetTM reagents, resulting in weak staining and results which are difficult of reproducing.

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

    5. Add 2 mL (or 1 volume) of EnzMetTM 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 EnzMetTM 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 EnzMetTM 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.

    For Western and other blotting applications, you should use EnzMetTM EnzMet Western Blot HRP Detection Kit (catalog number 6002) from Nanoprobes. Complete product information, instructions and protocols for this reagent are 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.

    More information:

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    Nanoprobes Receives New Grant

    Nanoprobes has received Research grant from the National Institute of General Medical Sciences (NIH) to continue the development of new probes for high-resolution electron microscopic labeling. The grant will support further development of the core gold nanoparticle preparation and chemical functionalization technologies that may enable further improvements to our product line, or to opportunities for new product development. The grant will also support research into new targeting methods to direct gold labels to proteins and other biological entities using fusion tags and other high-resolution labeling technologies.

    To help with this work, we welcome back Dr. John Dubendorff as a Research Scientist with a focus on protein expression and tagging technologies, and also welcome Luping Qian as a Research Technician who will assist with various aspects of the development of the gold labeling technology.

    More information:

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    Other Recent Publications

    The use of FluoroNanogold to check immunolabeling before TEM preparation was demnstrated once more by Szakmary and colleagues in their recent paper in the Journal of Cell Biology to investigate the function of Yb protein in Drosophila melanogaster. Yb regulates the proliferation of both germline and somatic stem cells in the Drosophila ovary by activating piwi and hh expression in niche cells. Alexa Fluor® 488 FluoroNanogold combined with an anti-Flag primary antibody was used to localize a Yb-Flag fusion protein.

    Ovaries were dissected from 5x Flag-Yb transgenic flies, fixed, and stained with mouse monoclonal anti-Flag M2 antibodies. Anti-mouse Alexa Fluor® 488 FluoroNanogold was used for secondary labeling (1:3 dilution). Ovaries were washed, lightly cross-linked with 0.1% glutaraldehyde, and the aldehyde quenched with 50 mM glycine in PBS. Several ovaries were directly inspected by immunofluorescence. Nanogold labeling of anti-Flag-Yb in the remaining ovaries was then enhanced using GoldEnhance. Subsequently, ovaries were rinsed and fixed with 2% glutaraldehyde in MOPS-buffered mammalian ringer solution for 1 hour at room temperature, then rinsed in same buffer and then in 0.1 MPO4 and 10 mM MgCl2, pH 6.1, at 4°C. Secondary fixation was performed in 1% OsO4 in the same phosphate buffer for 1 hour on ice. After a water rinse, 2% aqueous uranyl acetate was used as a block stain for 30 minutes, also on ice. After a water rinse, tissue was dehydrated in a graded 50-100% ethanol series and infiltrated in Araldite 506 epoxy resin. Germaria and attached early egg chambers were separated from the ovary by microdissection, oriented in puddles of Araldite on sheets of polyethylene, and allowed to partially polymerize to maintain the orientation. Araldite-filled BEEM capsules were inverted over the germaria. Several intact ovaries were also embedded. Curing was performed for 48 hours at 65°C. 0.5-µm sections were used to guide orientation, and 50 nm thin sections cut with a diamond knife and mounted on thin carbon films on copper grids. These sections were stained with aqueous 2% KmnO4, rinsed with Pal's bleach and water, and stained with Sato lead. Montages of the entire germaria were assembled from low magnification (3,300x) electron micrographs, and the clusters of gold labeling in individual cells were recorded at 10,000-12,000x.

    Yb protein was localized as discrete cytoplasmic spots exclusively in the somatic cells of the ovary and testis. These spots, which are different from all known cytoplasmic structures in D. melanogaster, are evenly electron-dense spheres, 1.5 µm in diameter, dubbed the Yb body): this was frequently associated with mitochondria and a less electron-dense, apparently RNA-rich sphere of similar size. One to two Yb bodies occur in each cell, and they are often located close to germline cells. The N-terminal region of Yb is required for hh expression in niche cells, whereas the C-terminal region is required for localization to Yb bodies. The entire Yb protein is necessary for piwi expression in niche cells. A double mutant of Yb and a novel locus show male germline loss, indicating that Yb has a role in maintaining male germline stem cells.

    Reference:

    • Szakmary, A.; Reedy, M.; Qi, H., and Lin, H.: The Yb protein defines a novel organelle and regulates male germline stem cell self-renewal in Drosophila melanogaster. J. Cell Biol., 185, 613-627 (2009).

    Cai and co-workers reported an immunoassay with resonance Rayleigh scattering detection in a recent issue of Sensors and Actuators B: Chemical. Functionalized gold nanoparticle probes coupling with resonance Rayleigh scattering (RRS) were developed for the picomolar detection of transferrin. This assay relies on the specific immune recognition of gold particles modified with anti-transferrin antibody; this was attached to the gold nanoparticle surface through cysteamine bioconjugation. Transmission electron microscopy (TEM), laser light scattering and UVvisible absorption spectroscopy were employed to construct and characterize the nanostructures and spectroscopic characteristics of the probes, and the experimental conditions, including conjugate concentration, reaction temperature, incubation time, pH value, salt concentration and coexisting substances, were optimized. The combination of the signal amplification provided by the gold probes with the high sensitivity of resonance Ralyleigh scattering (RRS) technique allow the detection limit of transferrin to reach 85 pM, and the linear detection range from 85 pM to 3.4 nM. The method was successfully applied to detect transferrin of human serum samples with good reproducibility, and it has potential for use in clinical diagnosis.

    Reference:

    • Cai, H.-H.; Yang, P.-H.; Feng, J., and Cai, J.: Immunoassay detection using functionalized gold nanoparticle probes coupled with resonance Rayleigh scattering Sens. Act. B, 135, 603-609 (2009).

    Another novel application of gold nanoparticles was described in a recent Nano Letters article by Wang and co-workers: as a contrast agent for photoaccoustic imaging. To detect macrophages in atherosclerotic plaques, plasmonic gold nanoparticles, 50 nm in diameter and passivated with coated with methoxy-polyethylene glycol-thiol, were introduced as a contrast agent for intravascular photoacoustic imaging. Phantom and ex vivo tissue studies showed that the individual spherical nanoparticles, resonant at 530 nm wavelength, produce a weak photoacoustic signal at 680 nm wavelength, while photoacoustic signal from nanoparticles internalized by macrophages is very strong due to the plasmon resonance coupling effect. This suggests that intravascular photoacoustic imaging can assess the macrophagemediated aggregation of nanoparticles and therefore identify the presence and the location of nanoparticles associated with macrophage-rich atherosclerotic plaques.

    Reference:

    • Wang, B.; Yantsen, E.; Larson, T.; Karpiouk, A. B.; Sethuraman, S.; Su, J. L.; Sokolov, K., and Emelianov, S. Y.: Plasmonic intravascular photoacoustic imaging for detection of macrophages in atherosclerotic plaques. Nano Lett., 9, 2212-2217 (2009).

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