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Updated: June 11, 2003

N A N O P R O B E S     E - N E W S

Vol. 4, No. 6          June 11, 2003


This monthly newsletter is to keep you informed about techniques to improve your immunogold labeling, highlight interesting articles and novel metal nanoparticle applications, and answer your questions. We hope you enjoy it and find it useful.

Have questions, or issues you would like to see addressed in the next issue? Let us know by e-mailing [email protected].

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Alexa Fluor Fluoronanogold: New Papers Demonstrate Advantages

John Robinson and Toshihiro Takizawa have pioneered the uses and applications of combined fluorescent and gold probes, and recently published two papers describing the use of combined Alexa Fluor®* 594 and Nanogold®-labeled streptavidin for correlative microscopy. The distribution of caveolin-1 in ultrathin cryosections of terminal villi of the human term placenta was determined by fluorescence and immunoelectron microscopy; the use of ultrathin cryosections enables high spatial resolution by fluorescence microscopy because there is essentially no out-of-focus fluorescence. The electron microscopy immunolabeling obtained with colloidal gold and FluoroNanogold were compared using a particle counting procedure: a higher number of particles was found with silver-enhanced FluoroNanogold than with colloidal gold.

Tissue was cut into small pieces and fixed in freshly prepared 4% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, containing 5% sucrose for 2 hr at room temperature. The samples were washed and embedded in 10% gelatin in the same buffer. The solidified gelatin was cut into smaller pieces and then cryoprotected by infiltration with 2.3 M sucrose in 0.1 M sodium cacodylate (pH 7.4) overnight at 4°C. Ultrathin cryosections (100-nm thickness or less) were cut on a Cryo P diamond knife (Diatome-US; Fort Washington, PA) and collected on droplets of 0.75% gelatin2.0 M sucrose or 1 % methylcellulose-1.15 M sucrose, then transferred to nickel Maxtaform finder grids (Graticules; Tonbridge, Kent, UK) to facilitate location of specific structures when going from the optical to the electron microscope. Pick-up solutions contained 0.05% sodium azide so that the cryosections could be stored until needed. Sections were immersed in a solution containing 1% non-fat dry milk and 5% fetal bovine serum in PBS (MFBSPBS) for 15 min at 37°C to remove the sucrose and gelatin, then washed three times in PBS and incubated in MFBSPBS with 0.05 % sodium azide to block nonspecific protein binding sites.

The grids were incubated with biotin-labeled goat anti-chicken (13 g/ml in MFBSPBS) for 30 min at 37°C, washed in PBS for 12 min with four changes, immersed in MFBSPBS, then incubated with Alexa Fluor 594 FluoroNanogold-streptavidin (diluted 1:50 in MFBSPBS) for 30 min at room temperature. The grids were then washed in PBS for 15 minutes with five changes and mounted on a glass microscope slide in PBS containing 1% N -propyl gallate and 50% glycerol, pH 8.0, to retard photobleaching, overlaid with an 18-mm round glass coverslip. The cryosections were examined immediately by optical microscopy and images were collected. The locations of regions of interest on the finder grid were noted for relocation in the electron microscope. The temporary slide preparations were then disassembled and the grids washed in PBS with five changes over 15 min. The ultrathin cryosections were then fixed in 2% glutaraldehyde in PBS for 30 min and washed in distilled water for 6 min with four changes. The sides of the grids opposite the sections were dried with filter paper. The grids were then floated on drops of distilled water and subsequently on drops of 50 mM 2-[N-morpholino]ethanesulfonic acid buffer, pH 6.15, for 4 min with two changes. FluoroNanogold bound to the cryosections was silver enhanced for 3 min to render them visible in the sections; a positive contrast enhancement procedure was used to visualize membrane profiles in the ultrathin cryosections. The same regions examined by fluorescence microscopy were relocated and electron micrographs collected.

References:

  • Takizawa, T., and Robinson, J. M.: Correlative Microscopy of Ultrathin Cryosections is a Powerful Tool for Placental Research. Placenta, 24, 557-565 (2003).
    • Abstract (courtesy of Science Direct, full text available):
  • Takizawa, T., and Robinson, J. M.: Ultrathin Cryosections. An important tool for immunofluorescence and correlative microscopy. J. Histochem. Cytochem., 51, 707-714 (2003).

More information:

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

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Optimizing Staining with Gold Enhancement

GoldEnhance can deliver excellent, clear signals in staining and labeling experiments. However, its differences from silver enhancement should also be noted. GoldEnhance typically develops faster: 1 minute is sufficient to enlarge Nanogold® particles to 3 nm, and three minutes is sufficient to enlarge them to 5 nm. 5-10 minutes is usually sufficient for full enlargement - to 20 nm or more in size. It is also important to note that the rate may be different - either faster or slower - in your system, and that getting the best result may require some trial-and-error.

If you find that development is too fast and you require more control, and shortening the development time is not an option, try the following:

  • Use more solution B. This moderates the reaction by stabilizing the gold source; by increasing the amount of B, the reaction may be slowed and better controlled. Therefore, it may be helpful to increase the ratio to 5 parts B to one part A.

  • Use a lower concentration of solution C. Since C contains the reducing agent, this will act to slow the reaction.

  • Lower the pH of your mixture; this acts to slow down the gold development and may compensate for any mechanism in your samples which accelerates it. Try washing the grid with 0.02 M sodium citrate buffer, pH 3.5, before applying GoldEnhance; alternatively, use hydrochloric acid to adjust the pH of solution D down to 5.5, then try the gold enhancement again.
More information:

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Applications of Nanogold®-Fab' Conjugates in Neuroscience

The specificity and resolution of Nanogold® conjugates make them highly effective for localizing and discriminating targets in tissues, and this is illustrated by two new studies. The sec6/8 (exocyst) complex is implicated in targeting of vesicles for regulated exocytosis in various cell types and is believed to play a role in synaptogenesis and brain development. Vik-Mo and colleagues used Nanogold-Fab' secondary antibody probes to localize the subunits sec6 and sec8 to neurons of adult rat brain, showing that immunoreactivity for the two subunits has a differential subcellular distribution. Sec6 is concentrated at the inside of the presynaptic plasma membrane in developing and mature neurons, while sec8 immunoreactivity shows a diffuse cytoplasmic distribution.

From rat-brain Vibratome sections, the hippocampus and cerebellar folia were dissected free and tissue samples incubated sequentially in the following solutions:

  1. TBST (Tris-buffered saline with Triton X-100) with 2 % human serum albumin (HSA).
  2. primary antibody diluted in TBST w/HSA for 48 h at 4°C.
  3. TBST w/HSA.
  4. Nanogold-Fab' 1:40 in TBST/HSA.
  5. TBST/HSA.
  6. PB (sodium phosphate buffer, pH 7.4).
  7. 2.0 % glutaraldehyde in PB.
  8. PB.
  9. HQ silver enhancement kit in dark room with safelight.
  10. Double-distilled water.
  11. PB.
  12. 0.5 % osmium tetroxide in PB.
  13. PB.
Sec6 was found to be transported along neurites on secretogranin II-positive vesicles, while sec6-negative/secretogranin II-positive vesicles stay in the cell body. The accumulation of sec6-positive vesicles at the plasma membrane at sites of cell cell contact, combined with the probably interaction of Sec8 with cytoskeletal elements, suggests that the Sec6/8 complex modulates exocytotic activity by targeting membrane material to its presynaptic destination.

Reference:

Vik-Mo, E. O.; Oltedal, L.; Hoivik, E. A.; Kleivdal, H.; Eidet, J., and Davanger, S.: Sec6 is localized to the plasma membrane of mature synaptic terminals and is transported with secretogranin ii-containing vesicles. Neuroscience, 119, 73-85 (2003).

Abstract (courtesy of Science Direct):
LINK

Meanwhile, Yu and group localized two variants of XIAP (X chromosome-linked inhibitor of apoptosis protein), which has been shown to inhibit cell death in a variety of cells, one full-length, the other with the carboxy-terminal RING domain, that can regulate cell death via protein degradation, deleted. Although both full-length and RING-deleted XIAP protected sympathetic neurons against death induced by nerve growth factor (NGF) withdrawal, electron microscopy with secondary Nanogold-Fab' conjugates showed that the two proteins were differentially localized in transfected neurons, with RING-deleted XIAP present in the cytoplasm and full-length XIAP found mostly in cytoplasmic protein aggregates. From their differing protective abilities and the lack of degradation and ubiquitination of the RING-deleted variant in neuroblastoma cells, the authors concluded that the RING domain was required for proteasomal association and ubiquitination of XIAP.

Cells were fixed with PLP fixative (2% formaldehyde, 0.01 M periodate, 0.075 M lysineHCl in 0.075 M phosphate buffer, pH 7.4) for 2 h at room temperature, permeabilized with 0.01% saponin, and immunolabeled using affinity-purified rabbit anti-human/mouse XIAP antiserum and anti-rabbit Nanogold-Fab, followed by silver-enhancement for two minutes with HQ Silver and gold-toning with 0.05% gold chloride. After being washed the cells were postfixed with 1% reduced osmium tetroxide for 1 h, and processed for Epon embedding.

Reference:

Yu, L. Y.; Korhonen, L.; Martinez, R.; Jokitalo, E.; Chen, Y.; Arumae, U., and Lindholm, D.: Regulation of sympathetic neuron and neuroblastoma cell death by XIAP and its association with proteasomes in neural cells. Mol. Cell Neurosci., 22, 308-318 (2003).

Abstract (courtesy of Science Direct):
LINK

More information:

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Smaller Immunoreactive Gold Conjugates

Thought Nanogold®-Fab' was the smallest gold immunoprobe you could get? Nanogold® labeling reagents may be used to prepare even smaller gold-labeled antibody fragments. Malecki and co-workers have described the bioengineering of recombinant single-chain variable fragment (scFv) antibodies and their derivatives containing metal-binding domains. As tested with surface plasmon resonance and enzyme-linked immunosorbent assay, affinity binding constants of the scFv (5.21 x 106 M-1) and scFv:MBD (4.17 x 106 M-1) were close to those of Fab proteolytic fragments (9.78 x106 M-1) derived from the parental IgG antibodies. A cysteine-modified form was labeled with Monomaleimido Nanogold via a cysteine residue. Nanogold-labeled anti-biotin Fab' and scFv fragments were then compared as secondary probes against a biotinylated primary against Pex3 to label peroxisomes in yeast cells: the scFv resulted in a much heavier specific labeling and label-free background.

Reference:

Malecki, M.; Hsu, A.; Truong, L., and Sanchez, S: Molecular immunolabeling with recombinant single-chain variable fragment (scFv) antibodies designed with metal-binding domains; Proc. Natl. Acad. Sci. USA, 99, 213-218 (2002).

Abstract (courtesy of the National Academy of Sciences of the USA):
http://www.pnas.org/cgi/content/abstract/99/1/213

Reprint (courtesy of the National Academy of Sciences of the USA):
http://www.pnas.org/cgi/reprint/99/1/213.pdf

Ribrioux and co-workers have also used fluorescent and Nanogold-labeled Fv fragments to demonstrate enhanced penetration and labeling. Reference:

Ribrioux, S., Kleymann, G., Haase, W., Heitmann, K., Ostermeier, C., and Michel, H.: Use of Nanogold- and Fluorescent-labeled Antibody Fv Fragments in Immunocytochemistry. J. Histochem. Cytochem., 44, 207-213 (1996).

Abstract (courtesy of the Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/44/3/207

Protein engineering has been proposed as a method for further improving this method. Jensen and Kornberg have proposed the use of ScFv fragments engineered to hold gold particles in a rigid geometry as a method for preparing gold probes with even higher resolution, making them useful for crystallographic structure determination methods as well as electron microscopy.

Reference:

Jensen, G. J., and Kornberg, R. D.: Single-particle selection and alignment with heavy atom cluster-antibody conjugates. Proc. Natl. Acad. Sci. USA, 95, 9262-9267 (1998).

Abstract (courtesy of the National Academy of Sciences of the USA):
http://www.pnas.org/cgi/content/abstract/95/16/9262

Reprint (courtesy of the National Academy of Sciences of the USA):
http://www.pnas.org/cgi/reprint/95/16/9262.pdf

More information:

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Other Recent Publications

Barth and group report a new staining procedure combining TUNEL methodology with pre-embedding immunogold labeling to detect DNA double-strand breaks in individual cells by electron microscopy and assess the accompanying ultrastructural alterations, to address concerns that the current cerminal transferase-mediated dUTP nick-end labeling (TUNEL) procedure does not label apoptotic alterations exclusively. The new staining procedure comprises (i) TUNEL staining of free-floating vibratome sections using fluorescein isothiocyanate (FITC)-labeled UTP, (ii) conversion of the fluorescence signal into an electron-dense signal using an anti-FITC antibody coupled with ultrasmall (0.8 nm) gold particles (Aurion) followed by silver enhancement (Nanoprobes HQ Silver), and (iii) osmication, embedding in Spurr resin and cutting of ultrathin sections.

Reference:

Barth, M.; Oulmi, Y.; Ehrenreich, H., and Schilling, L.: Pre-embedding immunogold labeling of TUNEL stain enables evaluation of DNA strand breaks and ultrastructural alterations in individual cells of neuronal tissue. Acta Neuropathol. (Berl), 104, 621-636 (2002).

Abstract (courtesy of Springer-Verlag):
LINK

Gutekunst and co-workers used a pre-embedding colloidal gold / enzymatic double labeling procedure to label HAP1 and LR11 for electron microscopy in rat and mouse brain tissue. Notably, they performed silver enhancement after DAB development, showing that DAB does not deposit silver and widening the options for users of this method. Sections were rinsed in PBS, blocked and incubated in a combination of HAP1B-C (1:1000) and mLR11 (1:100) overnight, then rinsed in PBS and incubated in a combination of biotinylated donkey anti-rabbit and ultrasmall colloidal gold-conjugated secondary antibody (Aurion) overnight. Sections were rinsed, incubated in avidinbiotin complex, and developed with DAB; then, after postfixation with 2.5% glutaraldehyde, gold particles in sections were intensified using silver enhancement (R-gent SE-EM kit, Aurion). Sections were then further fixed with 0.5% osmium tetroxide in 0.1 M PB for 15 min and processed for electron microscopy.

Reference:

Gutekunst, C. A.; Torre, E. R.; Sheng, Z.; Yi, H.; Coleman, S. H.; Riedel, I. B., and Bujo, H.: Stigmoid Bodies Contain Type I Receptor Proteins SorLA/LR11 and Sortilin. New perspectives on their function. J. Histochem. Cytochem., 51, 841-852 (2003).

Abstract (courtesy of the Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/51/6/841

DNA aptamers - single-stranded DNA probes that bind to proteins and other cellular targets - are another small probe that offers potential advantages of higher penetration, high binding constants, and ability to localize a variety of targets. Stanlis and McIntosh tested aptamers targeting green fluorescent protein for cellular localization; although they encountered problems with background binding, they found that solubilization in methanol and acetone - solvents used for freeze substitution - allowed the aptamer to readily enter cells.

Reference:

Kristi K.H. Stanlis, K. K. H., and McIntosh, J. R.: Single-strand DNA Aptamers as Probes for Protein Localization in Cells. J. Histochem. Cytochem., 51, 797-808 (2003).

Abstract (courtesy of the Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/51/6/797

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