N A N O P R O B E S     E - N E W S

• Abstract (courtesy of the American Journal of Surgical Pathology).

• Abstract (courtesy of SpringerLink).

More information:

• Enzyme Metallography for correlative labeling (our Microscopy & Microanalysis 2004 abstract)
• Enzyme Metallography - a New staining method (our Microscopy & Microanalysis 2002 abstract)
• Product application special report: Nanogold in situ hybridization
• Other product and research applications

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Labeling other Functional Groups: Phosphates in Lipids

Options for conjugating Nanogold to this molecule are very limited, because the PI(3)P molecule does not have any reactive functional groups to which Nanogold may be readily conjugated. Thiols or primary aliphatic amines would be best; however, as we have explained in previous articles, cross-linking chemistry is also possible for carboxylic acids or hydroxyls, although these procedures are slightly more difficult. However, PI(3)P has none of these, and contains only three groups which can be readily modified in any way. Is it possible to label any of them with Nanogold?

Considering each group in turn:

• The phosphate group: This is the best choice for labeling, because cross-linking chemistry has already been described in the literature. The simplest and best-documented approach is to activate the phosphate group in the same manner as a carboxylic acid: either by EDC/Sulfo-NHS coupling, activating with EDC (1-ethyl-3,3'-dimethylaminopropyl carbodiimide) followed by sulfo-NHS, or by treatment with CDI (N,N'-carbonyldiimidazole) in DMF (dimethylformamide). In either case, you could then react the activated ester with Monoamino-Nanogold.

  Reference:

  Chu, B. C. F.; Kramer, F. R., and Orgel, L. E.: Synthesis of an amplifiable reporter RNA for bioassays. Nucleic Acids Res., 14, 5591-5603 (1986).

  • Abstract (courtesy of Nucleic Acids Research).
  • Reprint (PDF) (courtesy of PubMed Central).

  Note that there is another option besides direct reaction of the activated ester with Monoamino Nanogold. The authors react their activated ester with cystamine, which is cleavable to give a thiol group: an alternative approach for Nanogold labeling is use cystamine exactly as described, cleave, and label using Monomaleimido Nanogold. Both reactions are described in more detail in the technical help section of our web site.

• The double bonds: While modification of the double bonds is possible chemically, it is likely to be undesirable because modification of these groups may change the biological or liposomal behavior of the molecule.

• The carbonyl (C=O) group: However, because this is actually part of an ester linkage, reactivity will be difficult and most methods will involve cleavage of the ester group, so part of the PI(3)P will be lost. Two potentially useful reactions are the Wittig or Horner-Wadsworth-Emmons reactions; however, these generally do not work well with esters, and you will probably need to find a variation that is adapted to this functionality. If your experiment would work with a cleaved derivative, this may be an option.

The reactions used for phosphate group labeling are shown below:

One consideration in selecting functional group for labeling is whether the site participates in, or might hinder, the activity that you will be using the labeled probe to study; if so, then this may not be the best strategy. It is often preferable to conjugate Nanogold to the hydrophilic region of the molecule rather than the hydrophobic domain, both because Nanogold itself is hydrophilic, and also because the assembly of lipids into liposomes and other structures is mediated by the interactions of the hydrophobic domain: this activity would be impacted by the attachment of a large label at this site.

More information:

• Guide to Gold Cluster Labeling
• Technical help: Nanogold labeling reagents
• Labeling Other Functional Groups: Hydroxyls
• Labeling Other Functional Groups: Aldehydes and Ketones
• Labeling Other Functional Groups: Carboxylic Acids
• Gold Lipids: catalog and general information
• Gold liposomes: our paper from Microscopy and Microanalysis 1996

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Double and Triple Labeling with Silver-Enhanced Nanogold® and HRP

In order to test this hypothesis, triple-immunohistochemical studies were performed in the SDCN. Triple-immunofluorescence histochemical studies showed that some axon terminals in close association with NK1R-immunopositive (NK1R-ip) neurons in the SDCN were immunopositive for both glutamic acid decarboxylase (GAD) and glycine transporter 2 (GlyT2). Therefore, electron microscopic dual- and triple-immunohistochemistry was conducted for GAD/GlyT2, GAD/NK1R, GlyT2/NK1R, and GAD/GlyT2/NK1R.

Spinal cords from rats perfused transcardially with 100 ml of 0.01M phosphate-buffered saline (PBS, pH 7.4), followed by 500 ml of 0.1M phosphate buffer (PB, pH 7.4) containing 4% (w/v) paraformaldehyde, 0.05% (w/v) glutaraldehyde and 15% (v/v) saturated picric acid were removed and placed in 0.1MPB (pH 7.4) containing 30% (w/v) sucrose overnight at 4°C. 50-micron frontal sections were collected consecutively in five series. After cryoprotection, sections were freeze-thawed in liquid nitrogen, placed in 0.05M Tris-buffered saline (TBS, pH 7.4) containing 20% normal goat serum (NGS) for 1 hour, then processed for electron-microscopic dual- and triple-immunohistochemistry. The first series of sections was used for double immunohistochemistry of GAD and GlyT2: these were incubated at room temperature sequentially with: (1) a mixture of 1 microgram/mL anti-GAD mouse IgG and 1 microgram/mL anti-GlyT2 guinea pig IgG for 24 hours; (2) a mixture of 10 microgram/mL biotinylated anti-[mouse IgG] donkey antibody (1:200) and Nanogold-IgG Goat anti-Guinea pig IgG (1:100).

The second and the third series were processed for dual immunohistochemistry for GAD/NK1R or GlyT2/NK1R, respectively. The immunoperoxidase method for either GAD or GlyT2 was combined with the immunogold-silver method for NK1R. The sections were incubated at room temperature sequentially for GAD/NK1R with: (1) a mixture of 1 microgram/mL anti-GAD mouse IgG and 1 microgram/mL anti-NK1R rabbit IgG for 24 hours; and (2) a mixture of biotinylated anti-[mouse IgG] donkey antibody and Nanogold-IgG goat anti-rabbit IgG; for GlyT2/NK1R with: (1) a mixture of 1 microgram/mL anti-GlyT2 guinea pig IgG (Chemicon) and 1 microgram/mL anti-NK1R rabbit IgG for 24 hours, and (2) with a mixture of biotinylated anti-[guinea pig IgG] goat antibody (Vector) and Nanogold-IgG goat anti-rabbit IgG for 1618 hours. The fourth series of sections was processed for triple-immunohistochemistry for GAD/GlyT2/NK1R: the immunoperoxidase method for GAD was combined with the immunogold-silver method for GlyT2 and NK1R. The sections were incubated at room temperature sequentially with: (1) a mixture of 1 microgram/mL anti-GAD mouse IgG, 1 microgram/mL anti- GlyT2 guinea pig IgG and 1 microgram/mL anti-NK1R rabbit IgG for 24 h; and (2) a mixture of biotinylated anti-[mouse IgG] donkey antibody, Nanogold-IgG Goat anti-Guinea pig IgG and Nanogold-IgG anti-rabbit IgG for 1618 hours. Between steps (1) and (2), the sections were washed with 0.05M TBS. The incubation medium for all antibodies was 0.05M TBS containing 2% (v/v) normal goat serum (NGS). Subsequently, the sections were processed for EM by (1) silver enhancement with HQ Silver; (2) incubation with ABC Elite Kit (1:50; Vector) and visualization of GAD-IR or GlyT2-IR using diaminobenzidine (DAB); (3) osmification with 1% OsO₄ in 0.1M PB (pH 7.4) for 1 hour; (4) counterstaining with uranyl acetate; and (5) flat embedding in Durcupan. Ultrathin sections through the SDCN were prepared, mounted on single-slot grids, and examined electron microscopically.

In the EM triple immunohistochemistry for GAD/GlyT2/NK1, both GlyT2-IR and NK1R-IR were marked by silver grains. However, in the EM dual immunochemistry for GlyT2/NK1, GlyT2-IR was detected only within axon terminals, while NK1-IR was observed specifically within profiles of neuronal cell bodies and dendrites; therefore, although represented by the same signal, the two targets could be distinguished in the EM triple GAD/GlyT2/NK1-immunohistochemistry: silver grains within the axon terminals and those within the profiles of neuronal cell bodies and dendrite were considered to represent GlyT2-IR or NK1-IR, respectively.

Axon terminals labeled with both electron-dense peroxidase immunoreaction products (GAD-ip) and silver grains (GlyT2-ip) were observed to be in symmetric synaptic contact with neuronal cell bodies or dendrites labeled with silver grains (NK1R-ip) within the SDCN. In total 45 synaptic axon terminals showing both GAD-IR and GlyT2-IR were observed upon NK1R-ip neuronal profiles within the SDCN; 39 (86.7%) of them were on dendritic profiles (Fig. 3D), and the remaining 6 (13.3%) on somatic profiles. On the basis of these results, it appears to be highly possible that some synaptic terminals upon NK1R-expressing SDCN neurons co-release GABA and Gly. The origins of the axon terminals co-expressing GAD- and GlyT2- immunoreactivity in the SDCN, however, still remain to be settled. The present results are in good accordance with the physiological data indicating that SDCN neurons might be modulated by presynaptic co-release of GABA and Gly.

Reference:

Feng, Y.-P.; Li, Y.-Q.; Wang, W.; Wu, S.-X.; Chen, T.; Shigemoto, R., and Mizuno, N.: Morphological evidence for GABA/glycine-cocontaining terminals in synaptic contact with neurokinin-1 receptor-expressing neurons in the sacral dorsal commissural nucleus of the rat. Neurosci. Lett., 18, 144-148.

• Abstract (courtesy of Science Direct)

References describing methods in more detail:

• Li, J. L.; Wang, D.; Kaneko, T.; Shigemoto, R.; Nomura, S., and Mizuno, N.: Relationship between neurokinin-1 receptor and substance P in the striatum: light and electron microscopic immunohistochemical study in the rat. J. Comp. Neurol., 418, 156163 (2000).
  • Abstract (courtesy of Wiley Interscience)

  Li, J. L.; Wu, S. X.; Tomioka, R.; Okamoto, K.; Nakamura, K.; Kaneko, T., and Mizuno, N.: Efferent and afferent connections of GABAergic neurons in the supratrigeminal and the intertrigeminal regions. An immunohistochemical tract-tracing study in the GAD67-GFP knockin mouse. Neurosci. Res., 51, 8191 (2005).

  • Abstract (courtesy of Science Direct)
  • Reprint (PDF) (courtesy of Science Direct)

More information:

• Silver-Nanogold and enzymatic double labeling: other references
• Technical help: silver enhancement
• Nanogold conjugates: catalog and general information
• Nanogold conjugates: instructions and product information
• Technical help: Nanogold antibody conjugates

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Keeping GoldEnhance Under Control

• Gold enhancement may safely be used before osmium tetroxide - it is not etched.
• May be used in physiological buffers (chlorides precipitate silver, but not gold).
• The metallographic reaction is less pH sensitive than that of silver.
• Gold gives a much stronger backscatter signal than silver.
• GoldEnhance is near neutral pH for best ultrastructural preservation.
• Low viscosity, so the components may be dispensed and mixed easily and accurately.

Alexander and co-workers show that gold enhancement works equally well with conventional colloidal gold in their recent paper in Proceedings of the National Academy of Sciences of the USA. They used a combination of immunofluorescence microscopy and SEM with gold-enhanced colloidal gold labeling to study the distribution and mobility of the sodium-hydrogen exchanger isoform 3 (NHE3), located on the apical membrane, which is fundamental to the maintenance of systemic volume and pH homeostasis. Proximal tubular reabsorption of filtered sodium is finely regulated by a variety of hormones and by changes in ionic composition and volume. The authors analyzed the subcellular distribution and dynamics of the exchangers in an epithelial line expressing NHE3 tagged with an exofacial epitope, which enabled monitoring exchanger mobility and traffic in intact cells.

Using determinations of fluorescence recovery after photobleaching in combination with dynamic measurements of subcellular distribution, NHE3 was found to exist in four distinct subcompartments in renal epithelial cells: a virtually immobile subpopulation retained on the apical membrane by interaction with the actin cytoskeleton, in a manner that depends on the sustained activity of Rho GTPases; a mobile subpopulation on the apical membrane, which can be readily internalized; and two intracellular compartments that can be differentiated by their rate of exchange with the apical pool of NHE3. The authors sought to disrupt the microvillar cytoskeleton in order to analyze its possible role in immobilizing NHE3, using K depletion and Clostridium difficile toxin B (TxB), an agent that impairs Rho GTPase function and has profound effects on microvillar structure. The results provide evidence that detachment of the immobile fraction from its cytoskeletal anchorage leads to rapid internalization.

For SEM, cells grown to confluence on 25-mm coverslips were fixed at 4°C with 4% paraformaldehyde for 30 minutes, washed, then labeled for 60 minutes with anti-HA antibody (1:500 dilution) in phosphate-buffered saline. Goat anti-mouse secondary antibody conjugated to 18 nm gold was then applied, followed by gold enhancement using GoldEnhance EM. Samples were dehydrated, mounted on stubs, coated with carbon, and then analyzed by using an XL30 environmental ESM (ESEM) (FEI). Images were generated through the combination of signals from the gaseous secondary electron detector and the backscatter electron detector. Analysis of particle distribution showed that 71% of the gold particles were associated with the apical membrane; however, treatment with TxB resulted in a drastic reduction in gold labeling, confirms that apical accumulation of NHE3 results, at least in part, from a retention process that involves Rho GTPases and the actin cytoskeleton.

These observations suggest that modulation of the mobile fraction of NHE3 on the apical membrane can alter the number of functional exchangers on the cell surface and hence the rate of transepithelial ion transport. Regulation of the interaction of NHE3 with the actin cytoskeleton can therefore provide a new mode of regulation of sodium and hydrogen transport.

Reference:

Alexander, R. T.; Furuya, W.; Szaszi, K.; Orlowski, J., and Grinstein, S.: Rho GTPases dictate the mobility of the Na/H exchanger NHE3 in epithelia: role in apical retention and targeting. Proc. Natl. Acad. Sci. USA, 102, 12253-12258 (2005).

• Abstract (courtesy of Proceedings of the National Academy of Sciences of the USA)

Depending on the sample properties and experimental conditions, the rate of gold particle enlargement with GoldEnhance can vary, and in some systems very rapid development of larger particles has been reported. If you observe the rapid formation of larger particles after a few minutes, we recommend the following:

1. Examine after a shorter development time. In some applications, 1-2 minutes may be enough to enlarge the particles to the desired size.

2. Substitute Solution D of the GoldEnhance with 0.05M sodium phosphate with 0.1M sodium chloride, adjusted to pH 5.5. This will reduce the pH of the reaction mixture, which will serve to slow down the rate of the gold deposition reaction.

3. Increase the sodium chloride concentration in your substitute for D to 0.5M. We have occasionally observed that higher ionic strengths reduces background, and may act to moderate the rate of development (possibly by shortening the range of ionic interactions in the solution).

4. Add a viscosity modifier, such as polyethylene glycol (PEG). A solution of one of the higher molecular weight forms, such as 1% carbowax (molecular weight 20,000), is best; dissolve 4% in solution D or your substitute; gum arabic, although it is somewhat less convenient to prepare since it requires longer to dissolve, is also effective. These are used to slow down development in silver enhancement and lead to uniform particle size and morphology, and it is reasonable to expect the same result with gold enhancement.

5. Adding a small amount of detergent, such as 0.1% Tween-20 (add as 0.4% in solution D or your substitute). Detergents are known to modify the growth of gold nanoparticles and may provide a growth constraint during gold enhancement.

The most efficient strategy is to start with the first two suggestions; if these do not give the desired results, work through the other three, (3), (4) and (5) in the order given.

More information:

• GoldEnhance - Catalog information
• Product information - GoldEnhance EM
• Gold enhancement: our Microscopy & Microanalysis 99 paper
• GoldEnhance - Technical help
• GoldEnhance - other references

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MSDSs Now Available Online

In addition, the product information files in PDF format have been updated or replaced. All fonts are now embedded - so the display problems reported on some platforms with the previous versions have been fixed.

More information:

• MSDS index page
• Product information index
• Catalog - master index
• Search page

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Other Recent Publications

Reference:

Kollman, J. M., and Quispe, J.: The 17Å structure of the 420 kDa lobster clottable protein by single particle reconstruction from cryoelectron micrographs. J. Struct. Biol., 151, 306-314 (2005).

• Abstract (courtesy of Science Direct)

Our pre-embedding immunoelectron microscopy customers continue to go strong. Reporting in the current Journal of Histochemistry and Cytochemistry, Sakulsak and co-workers used this method to study the ultrastructural distribution of a novel 503 amino-acid protein named SLAMP (sublingual acinar membrane protein) obtained by cloning a rat gene and expressed primarily in the sublingual gland. SLAMP is a novel lectin with 63% homology with human ERGIC-53-like protein, in the family of animal L-type lectins. The expression and localization of SLAMP in major rat organs and tissues was examined using a cDNA probe for SLAMP mRNA and rabbit antisera against SLAMP; abundant expression of SLAMP was demonstrated predominantly in the sublingual gland, with single sizes of the mRNA and protein 1.8 kb and 50 kDa, respectively, but not in other organs or tissues, including the parotid and submandibular glands. Enzyme immunohistochemistry showed that SLAMP was localized to the mucous acinar cells, but not to the serous demilunes or the duct system. For electron microscopic immunocytochemistry, cryostat sections on plastic slides, after successive pretreatments with 0.3% Tween 20 and 3% normal swine serum, were incubated with anti-SLAMP antisera at 1:1000 dilution overnight at room temperature, washed in phosphate-buffered saline (PBS) and incubated with Nanogold®-Fab' goat anti-rabbit at 1:100 dilution in PBS plus 0.5% BSA (PBS-BSA) for 1 hour. After washing with PBS-BSA, the sections were postfixed in 1% glutaraldehyde in PBS for 10 min, washed thoroughly in distilled water (DW) and developed with HQ silver for 5 minutes in the dark room. They were then washed in DW, postfixed with 0.5% osmium tetroxide for 15 minutes, washed and dipped in DW overnight at 4°C, then stained with 2% uranyl acetate. Finally, they were dehydrated in ethanol series and embedded in an epoxy resin. Ultrathin sections were made and examined in the electron microscope. SLAMP was localized predominantly to regions corresponding to the ER-Golgi intermediate compartment. Besides the sublingual gland, SLAMP immunoreactivity was also demonstrated in mucous cells of the minor salivary glands in oral cavity and of Brunners glands in the duodenum, suggesting that rat SLAMP plays a specific role in the early secretory pathway of glycoproteins in specific types of mucous cells.

Reference:

Sakulsak, N.; Wakayama, T.; Hipkaeo, W.; Yamamoto, M., and Iseki, S.: Cloning and characterization of a novel animal lectin expressed in the rat sublingual gland. J. Histochem. Cytochem., 53, 1335-1343 (2005).

• Abstract (courtesy of the Journal of Histochemistry and Cytochemistry).

Kloc and co-workers describe the use of a Nanogold-labeled anti-digoxigenin antibody, prepared by Nanogold labeling a comercially available antibody, for RNA in situ hybridization in their recent paper in Development. Light and electron microscopy of whole-mount in situ hybridization specimens. Organization of cytokeratin filaments, but not the actin cytoskeleton, depends on the presence of intact VegT mRNA and a noncoding RNA, Xlsirts. Destruction of either of these transcripts results in disruption of the cytokeratin cytoskeleton in a transcript-specific manner and interferes with proper formation of the germinal granules and subsequent germline development. In addition, analysis of the distribution of endogenous VegT and Xlsirts in live oocytes using molecular beacons showed that these RNAs are integrated into the cytokeratin cytoskeleton. These results demonstrate a novel structural role of coding and noncoding RNAs in the organization of the vegetal cortex of Xenopus oocytes.

Reference:

Kloc, M.; Wilk, K.; Vargas, D.; Shirato, Y.; Bilinski, S., and Etkin, L. D.; Potential structural role of non-coding and coding RNAs in the organization of the cytoskeleton at the vegetal cortex of Xenopus oocytes. Development, 132, 3445-3457 (2005).

• Abstract (courtesy of Development).

Details of preparation and method:

Kloc, M.; Bilinski, S.; Chan, A., and Etkin, L. D.: Mitochondrial ribosomal RNA. in the germinal granules in Xenopus embryos-revisited. Differentiation, 67, 8083 (2001).

• Abstract (courtesy of Blackwell-Synergy).

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