N A N O P R O B E S     E - N E W S

Vol. 10, No. 6          June 29, 2009

• 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.

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).
  • Abstract (courtesy of Oxford Journals).

• Paspalas, C. D., and Goldman-Rakic, P. S.: Microdomains for dopamine volume neurotransmission in primate prefrontal cortex. J. Neurosci., 24, 5292-5300 (2004).
  • Abstract (courtesy of the Journal of Neuroscience).
  • Reprint (courtesy of the Journal of Neuroscience).

• Paspalas, C. D., and Goldman-Rakic, P. S.: Presynaptic D1 dopamine receptors in primate prefrontal cortex: target-specific expression in the glutamatergic synapse. J. Neurosci., 25, 1260-1267 (2005).
  • Abstract (courtesy of the Journal of Neuroscience).
  • Reprint (courtesy of the Journal of Neuroscience).

More information:

• Nanogold conjugates: catalog information
• Nanogold-antibody conjugates: product information and instructions
• Gold enhancement: catalog and general information
• GoldEnhance EM: product information and intructions
• Technical help: Nanogold conjugates
• Technical help: Gold Enhancement
• References: Gold Enhancement

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Monofunctionality: does it Always Work?

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:

• Guide to Gold Cluster Labeling
• Nanogold labeling reagents: catalog information
• Monomaleimido Nanogold: instructions and product information
• Mono-Sulfo-NHS Nanogold: instructions and product information
• Technical help: Nanogold labeling reagents
• Catalog information: Nanogold labeling reagents
• Nanogold labeling references, sorted by application

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HIV Nef Protein Targeting Revealed by Nanogold®

• 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.

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).
  • Abstract (courtesy of the Journal of Virology).

More information:

• Nanogold conjugates: catalog information
• Nanogold-antibody conjugates: product information and instructions
• Nanogold-antibody conjugates: some other references
• Special feature: Nanogold pre-embedding labeling with Gold Enhancement
• Other references: Nanogold pre-embedding labeling
• Gold enhancement: catalog and general information
• GoldEnhance EM: product information and intructions
• Technical help: Gold Enhancement

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EnzMet^TM: Best for ISH and 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, EnzMet^TM 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:

• EnzMet^TM 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. EnzMet^TM is readily distinguished from all counterstains.
• Does not fade or bleach: can be archived indefinitely.

For clinical applications, EnzMet^TM 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 EnzMet^TM reagents from us for research use.

For in situ hybridization, use EnzMet^TM 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).
  • Abstract (courtesy of Science Direct).

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 EnzMet^TM are clearly visible.

EnzMet^TM 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: