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Updated: August 13, 2004

N A N O P R O B E S     E - N E W S

Vol. 5, No. 8          August 13, 2004

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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FluoroNanogold: Strategies for Fluorescent and Gold Labeling

Correlative fluorescent and immunogold labeling is a highly desirable, but challenging objective. Nanoprobes makes the process simpler with our FluoroNanogold probes, which include both the 1.4 nm Nanogold® label and either fluorescein, Alexa Fluor®* 488 or Alexa Fluor®* 594 covalently linked to Fab' fragments.

[Alexa Fluor® 488 FluoroNanogold: structure and fluorescence staining] (48k)]

Left: Structure of Alexa Fluor® 488 and Nanogold® - Fab', showing covalent attachment of components.

Right: Fluorescent staining obtained using combined combined Alexa Fluor® 488 and Nanogold® - Fab' tertiary probe. The specimen is a slide from the NOVA Lite ANA HEp-2 test, an indirect immunofluorescent test system for the screening and semi-quantitative determination of anti-nuclear antibodies (ANA) in human serum (see ). The slide was stained using positive pattern control human sera, a Mouse anti-Human secondary antiboidy, and combined Alexa Fluor® 488 and Nanogold® - Fab' tertiary probe. Specimens were washed with PBS (30 minutes) between each step, then blocked by the addition of 7 % nonfat dried milk to the tertiary antibody solution (original magnification 400 X).

Since immunofluorescent and immunogold labeling may work best under different conditions, combining fluorescence and gold labeling sometimes requires a degree of compromise between the optimum conditions for each label. The following methods may help to increase labeling sensitivity, and decrease background staining, with these probes.

  • For increased fluorescence, consider using the new Alexa Fluor conjugates rather than the fluorescein ones. Rather than fluorescein, these use bright, highly stable Alexa Fluor®* 488 and 594 fluorophores developed by Molecular Probes. Alexa Fluor®* 488 uses the same filter set as fluorescein, so you do not need to change your observation procedure. With Alexa Fluor® 594, you can differentiate a FluoroNanogold-labeled target from a second target labeled with fluorescein, Alexa Fluor® 488, green fluorescent protein, or other fluorophores. These new combined fluorescent and gold probes offer new performance levels and additional features:

    • Increased fluorescence brightness and higher quantum yield.
    • Improved solubility: lower background signal and higher signal-to-noise ratios.
    • Fluorescence remains high and consistent across a wider pH range.
    • Uses fluorescein (Alexa Fluor®* 488) or Texas Red (Alexa Fluor®* 594) filter sets.

  • For lowest background fluorescence, try blocking with 5% nonfat dried milk. This was also found to be particularly effective when 1 to 5% nonfat dried milk was mixed with the incubation buffer and added to the specimen with the FluoroNanogold conjugate. Cold-water fish gelatin may also be helpful.

  • Another way to increase signal is to use a tertiary labeling method: use your primary antibody, then detect with a biotinylated secondary antibody followed by FluoroNanogold-streptavidin. Tertiary labeling systems have been found to increase detection sensitivity in immunogold detection.

  • To obtain the cleanest gold labeling in the electron microscope, use a sodium citrate buffer wash after FluoroNanogold application, but before silver enhancement. 0.02 M sodium citrate at pH 7.0 works well with HQ Silver, while pH 3.5 works best with the Danscher silver formulation.

    Reducing hydrophobic interactions. Both the gold and fluorescent labels have some hydrophobicity: therefore, including in the wash buffer agents that reduce hydrophobic interactions may help to reduce non-specific binding. Suitable reagents:

    • 0.6 M triethylammonium bicarbonate buffer (prepared by bubbling carbon dioxide into an aqueous suspension of triethylamine with stirring: see Safer, D.; Bolinger, L., and Leigh, J. S.: Undecagold clusters for site-specific labeling of biological macromolecules: simplified preparation and model applications. J. Inorg. Biochem., 26, 77 (1986));
    • 0.1 to 1% detergent, such as Tween-20, or Triton X-100;
    • 0.1 to 0.5% of an amphiphile, such as benzamidine or 1,2,3-trihydroxyheptane.
The following references also provide helpful protocols for labeling using FluoroNanogold:

  • Takizawa, T., and Robinson, J. M.: Correlative Microscopy of Ultrathin Cryosections is a Powerful Tool for Placental Research. Placenta, 24, 557-565 (2003).

  • Takizawa, T., and Robinson, J. M.: Ultrathin Cryosections. An important tool for immunofluorescence and correlative microscopy. J. Histochem. Cytochem., 51, 707-714 (2003).

Although it would be highly desirable to prepare combined fluorescent and gold probes using larger colloidal gold particles, the fluorescence quenching properties of gold particles larger than about 3 nm results in almost total loss of fluorescence if these are linked to the same antibody. Gold particles are excellent acceptors for fluorescence resonance energy transfer, or FRET, and absorb strongly at the emission wavelengths of commonly used fluorophores, as we have described:

  • Powell, R. D.; Halsey, C. M. R., and Hainfeld, J. F.: Combined fluorescent and gold immunoprobes: Reagents and methods for correlative light and electron microscopy. Microsc. Res. Tech., 42, 2-12 (1998).

In order for fluorescence to be retained, the fluorescent tag has to be positioned far enough away from the gold particle that a significant fraction is emitted directly rather than lost through energy transfer. Nanogold is sufficiently small that fluorescence is preserved in such conjugates, but with larger gold conjugates, fluorescence is almost completely quenched.

A solution is possible for combined 6 nm gold and fluorescent labeling: use a tertiary system in which the secondary antibody is gold-labeled, and the tertiary antibody is fluorescently labeled. In this way the resolution of the secondary gold label is preserved, but the fluorophore is now far enough away from the gold that usable fluorescence is preserved. This has been described by Kandela and co-workers in papers presented at Microscopy and Microanalysis 2003 and Microscopy and Microanalysis 2004:

  • Kandela, I. K.; Meyer, D. A.; Oshel, P. E.; Rosa-Molinar, E., and Albrecht, R. M.: Fluorescence Quenching by Colloidal Heavy Metals: Implications for Correlative Fluorescence and Electron Microscopy Studies. Microsc. Microanal., 9, (Suppl. 2: Proceedings) (Proceedings of Microscopy and Microanalysis 2003); Piston, D.; Bruley, J.; Anderson, I. M.; Kotula, P.; Solorzano, G.; Lockley, A., and McKernan, S., Eds.; Cambridge University Press, New York, NY, 1194CD (2003).

  • Kandela, I. K.; Bleher, R., and Albrecht, R. M.: Correlative Labeling Studies In Light and Electron Microscopy. Microsc. Microanal., 10, (Suppl. 2: Proceedings) (Proceedings of Microscopy and Microanalysis 2004); Anderson, I. M.; Price, R.; Hall, E.; Clark, E., and McKernan, S., Eds.; Cambridge University Press, New York, NY, 1212CD (2004).

If you would like us to help find the most effective FluoroNanogold labeling protocol for your project, please contact our technical support people. We will be glad to advise.

More information:

*Alexa Fluor is a registered trademark of Molecular Probes, Inc.

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Nanogold® Used to Study Neuronal Glutamate Receptor Activation

Kushmerick and colleagues contribute the latest in a productive line of experiments using Nanogold® labeling to probe neurological features by electron microscopy. In conjunction with electrophysiological measurements and fluorescence confocal microscopy, they used immunoelectron microscopy to probe the role of activation of group I metabotropic glutamate receptors (mGluRs) and CB1 cannabinoid receptors (CB1Rs) in the inhibition of synaptic currents at the calyx of Held synapse in the medial nucleus of the trapezoid body (MNTB) of the rat auditory brainstem.

In about 50% of MNTB neurons, activation of group I mGluRs by the specific agonist (s)-3,5-dihydroxyphenylglycine (DHPG) reversibly inhibited both AMPA receptor- and NMDA receptor-mediated EPSCs to a similar extent, suggesting inhibition of glutamate release. A reversible reduction of Ca2+ currents by DHPG was found by presynaptic voltage-clamp experiments. In about 50% of the tested cells, the CB1 receptor agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN) also reversibly inhibited EPSCs, presynaptic Ca2+ currents, and exocytosis; in any given cell, inhibition by both reagents was similar. DHPG inhibition was blocked by the CB1R antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) and occluded by WIN, indicating that DHPG and WIN operate via a common pathway. Inhibition of EPSCs by DHPG, but not by WIN, was abolished after dialyzing 40 mM BAPTA into the postsynaptic cell, suggesting that DHPG activates postsynaptic mGluRs.

Cellular distribution of CB1Rs and mGluR1 was examined by confocal immunofluorescence and immunoperoxidase light microscopy. Postsynaptic mGluR1a immunoreactivity was localized opposite presynaptic CB1 at the calyx of Held. mGluR1a is restricted to the postsynaptic principal cell in the MNTB; comparison of Rab3 (a vesicle-associated protein that defines the calyx of Held terminal) immunofluorescence indicated that immunoreactivity to mGluR1a is concentrated at the plasma membrane of a majority of principal cells, but does not colocalize with Rab3a immunoreactivity. B, CB1 and mGluR1a are located on opposite sides of the calyceal synapse in the MNTB of a P14 rat. mGluR1 is located opposite Rab3a at the calyceal synapse, while CB1 and Rab3a are colocalized presynaptically in the calyx of Held terminal.

Electron microscopy was then used to find the subcellular localization of . Five Sprague Dawley rats from P14 were anesthetized and transcardially perfused at room temperature (20-25°C) with PBS, pH 7.4, for 20 sec, followed by 500 ml of ice-cold fixative containing 4% formaldehyde, 0.025% glutaraldehyde, and 0.2% picric acid in 0.1 M phosphate buffer (PB), pH 7.4, for 1015 min. Brainstem blocks were washed thoroughly in 0.1 M PB. Parasagittal vibratome sections (50 microns) containing MNTB were cut and collected in 0.1 M PB at room temperature, preincubated in 20% NGS/PBS for 1 hr at room temperature, and incubated with affinity-purified antisera (1 mg/ml in 1.5% NGS/PBS) recognizing mGluR1a or C terminus of the CB1 receptor for 3 days at 4°C. After several washes in PBS, tissue sections were incubated in Nanogold-Fab' goat anti-rabbit IgG diluted 1:100 in 1.5% NGS/PBS for 3 hr on a shaker at room temperature. The MNTB sections were then washed in PBS overnight, postfixed in 1% glutaraldehyde for 10 minutes, washed in double-distilled water, then silver intensified using HQ Silver for about 12 minutes. The silver-enhanced tissue sections were osmicated in 1% PB (0.1 M), pH 7.4, osmium tetroxide for 20 minutes on a shaker at room temperature, dehydrated in graded alcohols, transferred to propylene oxide, and embedded flat in Epon 812. Ultrathin sections were collected on mesh nickel grids and stained with lead citrate.

Presynaptic terminals of calyces of Held were immunoreactive for CB1, which was localized to presynaptic membrane specializations at calyx of Heldprincipal cell synapses. Statistical analysis indicated approximately threefold higher density of silver-intensified gold particles on membranes of calyces of Held than on the plasmalemma of principal cell bodies. Silver-intensified gold particles indicating the presence of mGluR1a were also distributed over membranes of principal cell bodies. Label for mGluR1a was markedly accumulated at perisynaptic sites of the numerous postsynaptic specializations of the calyciform synapses. Furthermore, immunoparticles were also localized to plasmalemmal portions not directly associated with presynaptic membranes, suggesting extrasynaptic mGluR1a receptors. Quantitative evaluation of immunoparticles confirmed the prevalent postsynaptic localization of mGluR1a: membranes of principal perikarya were immunolabeled approximately three times more frequently than the presynaptic membranes of calyces of Held. Thus, both group I mGluR and CB1 are expressed in the MNTB, with CB1 located primarily in the calyx and mGluR1a in MNTB principal neurons, consistent with the light microscope results. These data support a mechanism in which activation of postsynaptic mGluRs triggers the Ca2+-dependent release of endocannabinoids that activate CB1 receptors on the calyx terminal, which leads to a reduction of presynaptic Ca2+ current and glutamate release.


Kushmerick, C.; Price, G. D.; Taschenberger, H.; Puente, N.; Renden, R.; Wadiche, J. I.; Duvoisin, R. M.; Grandes, P., and von Gersdorff, H.: Retroinhibition of presynaptic Ca2+ currents by endocannabinoids released via postsynaptic mGluR activation at a calyx synapse. J. Neurosci., 24, 5955-5965 (2004).

More information:

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Engineered Viruses for Nanopatterning Gold Particles

Gold particles are important components for nanobiotechnology the convergence of nanotechnology with biological design. Their wide variety of optical and electronic properties give many potential nanotechnology applications. Furthermore, gold particles with chemically selective reactivity may be conjugated to specific sites within biological molecules where these properties impart useful functionality, providing a way to use the organizing capability of biological systems to exploit these properties. A new book, "Nanobiotechnology: Concepts, Applications and Perspectives," edited by Mirkin and Niemeyer, discusses this field and contains several chapters on the applications of gold particles, including many applications of our Nanogold® labeling technology.

"Nanobiotechnology: Concepts, Applications and Perspectives" - details and ordering information

Previously, we reported how McMillan and co-workers used biomolecule templating to fabricate nanoscale two-dimensional arrays of quantum dots. Gold and semiconductor nanoparticles were attached to crystalline arrays of hollow, double-ring protein structures prepared by the modification of one of the three subunit proteins of the heat shock protein HSP60 from Sulfolobus Shibatae, a bacterium that lives in geothermal hot springs at temperatures up to 85°C and pH2. Two classes of variants were made: in the first, a cysteine was engineered into a 28 amino acid loop that protrudes into the central cavity, giving a pore 3 nm in diameter. In the second, the loop was removed and the cysteine engineered into the apical domain itself to give an exposed, 9 nm cavity. Two-dimensional arrays of each, treated with different sized gold particles, produced different patterns. 5 and 10 nm gold bound to the apical thiols of both variants to give ordered arrays of gold particles, postulated to rest on top of the assembled protein complex, tethered by the thiols, rather than inside the hollow core. Cysteine-modified, loopless subunits, labeled using Monomaleimido Nanogold® and reassembled, were found to contain multiple Nanogold particles on the inner surface of the structure, and XEDS measurements suggested that each pore contained up to nine Nanogold particles.


  • Mogul, R.; McMillan, R. A.; Paavola, C. D.; Trent, J. D., and Zaluzec, N. J.: Self-Assembled 2D Protein Crystals as Templates for Ordered Metallic Nano-Arrays. Microsc. Microanal., 9, (Suppl. 2: Proceedings) (Proceedings of Microscopy and Microanalysis 2003); Piston, D.; Bruley, J.; Anderson, I. M.; Kotula, P.; Solorzano, G.; Lockley, A., and McKernan, S. (Eds); Cambridge University Press, New York, NY, p. CD274 (2003).

  • McMillan, R. A.; Paavola, C. D.; Howard, J.; Chan, S. L.; Zaluzec, N. J., and Trent, J. D.: Ordered nanoparticle arrays formed on engineered chaperonin protein templates. Nat. Mater., 1, 247-52 (2002).

Kim and co-workers extend the method to three dimensions with their report at Microscopy and Microanalysis 2004. They demonstrated the controlled 3-dimensional organization of 5nm gold nanoparticles at specific sites, using cysteine-modified mutants of the Cowpea Mosaic Virus (CPMV) as templates. The CPMV capsid is an icosahedron with a diameter of 28.4nm, formed by 60 identical copies of an asymmetrical subunit. Native CPMV virus contains no cysteines on the exterior capsid surface: engineering in cysteines at selected points was used to provide selective attachment sites for gold nanoparticles of different sizes. In this study, two different cysteine-containing mutants, designated BC and EF, were used to produce two different patterns of cysteines and attached gold nanoparticles on the CPMV capsid; the observed pattern in the transmission electron microscope showed that the gold particle locations matched up very well with the position of the cysteines in the structural model. Clear differences in gold distribution were shown for the different CPMV mutants, demonstrating that the gold particles bind specifically to the cysteine thiols rather than non-specifically to the capsid outer surface; close packing of up to 13 5 nm gold particles per capsid was observed, and the virus particles could be individually manipulated. Electrical characterization is promised.

Cowpea Mosaic Virus (CPMV) is highly suited to such studies, since it is easily engineered and prepared in relatively large quantities. Monomaleimido Nanogold provides another method for labeling modified CPVM, previously described by Wang and group. Nanogold labeling, however, is not restricted to thiols: using Mono-Sulfo-NHS-Nanogold, you can also label at primary amines.


  • Kim, M. J.; in het Panhuis, M.; Gupta, R.; Blum, A. S.; Ratna B. R.; Gnade, B. E., and Wallace, R. M.: Nano-patterning and Manipulation of Genetically Engineered Virus Nanoblocks. Microsc. Microanal., 10, (Suppl. 2: Proceedings) (Proceedings of Microscopy and Microanalysis 2004); Anderson, I. M.; Price, R.; Hall, E.; Clark, E., and McKernan, S., Eds.; Cambridge University Press, New York, NY, 26 (2004).

  • Wang, Q.; Lin, T.; Johnson, J. E.; and Finn, M. G.: Natural supramolecular building blocks. Cysteine-added mutants of cowpea mosaic virus. Chem. Biol., 9, 813-819 (2002).

More information:

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Laser-Activated Capsules of Silver Nanoparticles

Skirtach and group report another novel observation with potential nanotechnology implications in the current Langmuir: they demonstrated the release of either silver nanoparticles or an infrared dye, encapsulated within poly(sodium 4-styrenesulfonate) (PSS: ~70 kDa), or poly(allylamine hydrochloride) (PAH: 50-60 kDa), using a laser to cut the polyelectrolyte capsules and release the contents.

Polyelectrolyte capsules containing silver nanoparticles were prepared by Tollens probe reaction (silver mirror reaction) on the surface of polyelectrolyte multilayer coated latexes. Precoating with polyelectrolyte multilayers was done by mixing 1800 microliters of 1.5% suspension of 5 micromolar MF latexes in 0.5 M NaCl (aqueous solution) with 200 microliters of 5 mg/mL PSS solution. After 15 minutes of incubation, triple washing with water and centrifugation, particles coated with one PSS layer reversed their charge from positive to negative and were coated with a PAH layer in a similar manner. This adsorption cycle was repeated until two (PSS/PAH) bilayers were formed. Next, the metallic layer was assembled onto the precoated latexes by mixing the suspension of the shells with 40 microliters of acetaldehyde. In an ultrasonic bath, 100 microliters of 5% freshly prepared [Ag(NH3)2]NO3 was added by slow dropwise addition. After 60 min the shells were washed by five centrifugation cycles and the coating made stable against desorption of silver particles by further coating with two additional (PSS/PAH) bilayers. Hollow capsules having the final (PSS/PAH)2)-Ag-(PSS/PAH)2) structure were obtained by dissolution of MF cores in 0.1 M HCl and cleaning by five centrifugation cycles. Capsules containing the infrared dye IR-806 were produced by layer-by-layer adsorption where PAH was used as a polycation and the IR-806 dye as a low-molecular weight anion, using 16 micrometer CaCO3 particles as templates.

A CW laser diode at 830 nm with optical power up to 80 mW was used in the experiments. The collimated laser beam was focused onto the sample through a microscope objective (100X magnification, N.A.1.25). The sample was positioned in the field of view by a micrometer resolution XYZ stage and illumination made in transmission mode using a 150 W white light source, and images recorded using a CCD camera. The solution containing the capsules was deposited onto a microscope slide under the microscope objective. Ablation, cutting and deformation of the silver nanoparticle-loaded capsules was observed at laser powers up to 25mW, attributed to direct local heating of the silver nanoparticles; the infrared dye required laser power of 60 mW or greater for activation, attributed to a different heating mechanism: electronic-vibronic-phonon energy conversion that induces heating.


Skirtach, A. G.; Antipov, A. A.; Shchukin, D. G., and Sukhorukov, G. B.: Remote Activation of Capsules Containing Ag Nanoparticles and IR Dye by Laser Light. Langmuir, 20, 6988-6992 (2004).

Gold Lipids, another novel gold particle conjugate available from Nanoprobes, may also be used to insert metal nanoparticles into organic layers, in this case liposomes; heavily gold-decorated liposomes have been observed by scanning transmission electron microscopy, and the gold-decorated liposomes can adopt several different morphologies. Adler-Moore also used these as probes to label a liposomal antifungal drug in order to study its interaction with components of fungal cells.


  • Hainfeld, J. F.; Furuya, F. R., and Powell, R. D.: Metallosomes. J. Struct. Biol., 127, 152-160 (1999).

  • Adler-Moore, J.: AmBisome targeting to fungal infections. Bone Marrow Transplantation, 14, S3-S7 (1994).

More information:

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Other Recent Publications

Lucocq and co-workers report a simple, rapid method for estimating gold labeling over different cellular compartments and organelles in their paper in the current issue of the Journal of Histochemistry and Cytochemistry. The method uses a sampling approach in which a regular array of microscopic fields or linear scans is positioned randomly on labeled sections. Gold particles (in this case 5 and 16 nm) from these were counted and assigned to identifiable cell structures, then used to construct a gold labeling frequency distribution of the labeled compartments. For ultrathin cryosections labeled for a range of different proteins and for a signaling lipid, it was found by scanning labeled sections at the electron microscope that counting 100200 particles on each of two grids yields a reproducible and rapid assessment of the pattern of labeling proportions over 1016 compartments. More precise estimates require counting 100200 particles over each compartment of interest.


Lucocq J. M.; Habermann A.; Watt S.; Backer J. M.; Mayhew T. M., and Griffiths G.: A rapid method for assessing the distribution of gold labeling on thin sections. J. Histochem. Cytochem., 52, 991-1000 (2004).

Confirmation that HQ Silver works just as well with gold probes from other sources as it does with Nanogold® was provided by Nio and group, who used it to enhance a small (1 nm) colloidal gold secondary probe targeted to the chitinase family protein Ym1 in 15 micrometer mouse spleen sections. Comparison with in situ hybridization results showed that the monoclonal antibody directed to YM1 also localized its isoform Ym2. Ym1 was principally expressed in the lung, spleen, and bone marrow: Ym1-expressing cells in the lung were alveolar macrophages, and immunoreactivity for Ym1 was localized in rough endoplasmic reticulum. Ym1-expressing cells in the spleen gathered in the red pulp and were electron microscopically identified as immature neutrophils; and in the bone marrow, immature neutrophils were intensely immunoreactive, but lost this immunoreactivity with maturation. Needle-shaped crystals in the cytoplasm of macrophages, which formed erythroblastic islands, also showed intense Ym1 immunoreactivity. Ym2 was found in the stomach, and Ym2 expression was restricted to the stratified squamous epithelium in the junctional region between forestomach and glandular stomach.


Nio, J.; Fujimoto, W.; Konno, A.; Kon, Y.; Owhashi, M., and Iwanaga, T.: Cellular expression of murine Ym1 and Ym2, chitinase family proteins, as revealed by in situ hybridization and immunohistochemistry. Histochem. Cell Biol., 21, 473-82 (2004).

Bilinksi and Kloc demonstrate the same thing for LI Silver in their recent Experimental Cell Research paper, and also provide some useful protocols for both immunolabeling and whole mount in situ hybridization for electron microscopy: using these methods, they show that the spliceosomal Sm proteins, Xcat2 mRNA, and DEADbox RNA helicase XVLG1, are present in the perinuclear electron-dense material called the "nuage," a conserved feature of germ cells in many animal species believed to be a precursor of germinal (or polar or P) granules. The whole mount in situ hybridization procedure used a very small gold-labeled anti-digoxigenin antibody to bind antisense digoxigenin labeled RNA probes for Xcat2 and DEADSouth, in Xenopus laevis oocytes. Immunostaining was conducted using primary monoclonal antibodies followed by 1 nm gold-labeled secondaries in ultrathin Lowicryl-embedded sections; some procedures were repeated in whole-mount preparations.


Bilinski, S. M.; Jaglarz, M. K.; Szymanska, B.; Etkin, L. D., and Kloc, M.: Sm proteins, the constituents of the spliceosome, are components of nuage and mitochondrial cement in Xenopus oocytes. Exp. Cell Res., 299, 171-178 (2004).

The whole mount in situ hybridization method for electron microscopy is described more fully in the following reference:

Kloc, M.; Bilinski, S.; Chan, A. P., and Etkin, L. D.: Mitochondrial ribosomal RNA in the germinal granules in Xenopus embryos revisited. Differentiation, 67, 80-83 (2001).

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