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Updated: January 13, 2004

N A N O P R O B E S     E - N E W S

Vol. 5, No. 1          January 13, 2004


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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Nanogold® Resolves the Distribution of PECAM-1

Nanogold®-labeled Fab' fragments are the smallest commercially available immunogold probes suitable for TEM visualization, smaller than unlabeled IgG molecules, and therefore give both most qualitative labeling, and highest ability to access hindered or restricted antigens. Since they are smaller than a primary IgG, they may be used to demonstrate differences in labeling distribution obtained using different primary antibodies against the same target. Feng and co-workers used these advantages to resolve a debate over the distribution of platelet endothelial cell adhesion molecule (PECAM-1, CD31) in vascular endothelium. It was originally reported to be highly concentrated at interendothelial cell contacts; but recent studies have claimed that CD31 is distributed evenly over the entire endothelial cell surface. Feng and co-workers have re-investigated this question using two different murine anti-CD31 antibodies (MEC 13.3 and M-20), in a pre-embedding immunonanogold electron microscopic procedure that allows precise label quantitation.

Mice were sacrificed by CO2 narcosis and tissues were immediately fixed in 4% paraformaldehyde in 0.02 M PBS, pH 7.4, for 4 hr at room temperature (RT), washed in 0.02 M PBS, pH 7.4, and immersed overnight in 4°C 30% sucrose in 0.02 M PBS, pH 7.4, then embedded in OCT compound and stored in liquid nitrogen. 10-micrometer cryostat tissue sections were cut, collected on pre-cleaned glass slides, and air-dried for 20 minutes. Sections were prepared as follows at room temperature:

  1. Wash in 0.02 M PBS, pH 7.4 (5 min).
  2. Immerse in 50 mM glycine in 0.02 M PBS, pH 7.4 (10 min).
  3. Wash in 0.02 M PBS, pH 7.4 (5 min).
  4. Immerse in 5% normal goat serum (NGS) (Vector Laboratories; Burlingame, CA) (20 min).
  5. Incubate in the primary antibody (MEC 13.3, M-20, or C-19) at dilutions of 1:25, 1:50, or 1:100 in 0.02 M PBS (60 min).
  6. Wash in 0.02 M PBS, pH 7.4 (3 X 5 min).
  7. Incubate in Nanogold-Fab' goat anti-rat or rabbit anti-goat secondary antibody diluted 1:50 or 1:100 in 0.02 M PBS, pH 7.4, 60 min.
  8. Wash in 0.02 M PBS, pH 7.4 (3 X 5 min).
  9. Postfix in 1% glutaraldehyde in 0.02 M PBS, pH 7.4 (2 min).
  10. Wash in distilled water (3 X 5 min).
  11. Develop 6 min with HQ silver in the darkroom.
  12. Wash in distilled water (2 X 2 min each.
  13. Fix in 5% sodium thiosulfate (1 min).
  14. Wash in distilled water (3 X 5 min).
  15. Postfix in 1% osmium tetroxide in sym-collidine buffer, pH 7.4 (10 min).
  16. Wash in 0.05 M sodium maleate, pH 5.2 (5 min).
  17. Stain with 2% uranyl acetate in 0.05 M sodium maleate buffer, pH 6.0 (5 min).
  18. Rinse in distilled water (5 min).
  19. Dehydrate in graded ethanols and infiltrate with a propylene oxide-eponate (Ted Pella, Redding, CA).
  20. Embed by inverting eponate-filled plastic capsules over the slide-attached tissue sections.
  21. Polymerize at 60°C for 16 hr.
  22. Separate eponate blocks from glass slides by brief immersion in liquid nitrogen, cut thin sections with diamond knife and collect on uncoated 200-mesh copper grids.

Antibody against MEC 13.3 reacted strongly with the luminal and abluminal plasma membranes of the endothelial cells lining microvessels in normal tissues and in angiogenic vessels induced by a tumor and vascular endothelial growth factor (VEGF-A). Lateral plasma membranes were significantly less labeled. Conversely, M-20 strongly labeled the cytoplasmic face of the lateral plasma membranes, although sparing specialized junctions, and only weakly labeled the luminal and abluminal plasma membranes. Both antibodies stained a significant minority of vesicles and vacuoles comprising the vesiculovacuolar organelle (VVO). Neither was reactive in CD31-null mice. These results indicate that CD31 is distributed over the entire endothelial cell surface, except at specialized junctions, and in VVOs, but is not equally accessible to different antibodies in all locations.

Reference:

Feng, D.; Nagy, J. A.; Pyne, K.; Dvorak, H. F., and Dvorak, A. M.: Ultrastructural Localization of Platelet Endothelial Cell Adhesion Molecule (PECAM-1, CD31) in Vascular Endothelium. J. Histochem. Cytochem., 52, 87-102 (2004).

Abstract (courtesy of the Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/52/1/87

The preparation and labeling procedure is described in more detail in an earlier paper:

Feng, D.; Nagy, J. A.; Brekken, R. A.; Pettersson, A.; Manseau, E. J.; Pyne, K.; Mulligan, R.; Thorpe, P. E.; Dvorak, H. F.; and Dvorak, A. M.: Ultrastructural localization of the vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) receptor-2 (FLK-1, KDR) in normal mouse kidney and in the hyperpermeable vessels induced by VPF/VEGF-expressing tumors and adenoviral vectors. J. Histochem. Cytochem., 48, 545-556 (2000).

More information:

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Fluoronanogold shows Path of Antisense Oligonucleotides to Targets

In order to develop successful applications of antisense oligonucleotides (ODNs) in cell biology and therapy, a detailed understanding of the mechanism of antisense action is essential. Shi and group have employed phosphorothioate ODNs directed against specific regions of the mRNA of the serotonin 5HT1A receptor, formulated in cationic liposomes, to study the localization of antisense reactions and elucidate the mechanism. The biotinylated oligonucleotides were localized at the macromolecular level after checking fluorescence distribution by electron microscopy after labeling with FluoroNanogold-streptavidin, a unique probe containing both fluorescein and the 1.4 nm Nanogold® label covalently linked to streptavidin.

RN46 cells were grown to 70 % confluency. Antisense ODNs were complexed with sonicated cationic lipid vesicles consisting of SAINT-2 and DOPE (1:1). In preliminary work, Lipofectamine 2000, purchased from Invitrogen, was also employed, and antisense ODN complexes were prepared essentially as instructed by the manufacturer. Where indicated, 0.5 mol % N-(lissamine rhodamine sulfonyl)-PE (NRh-PE, Avanti Polar Lipids Inc) was included in the lipid mixture to monitor the fate of the lipid complex by confocal fluorescence microscopy. Antisense / cationic lipid complexes were prepared as follows: 15 nmol SAINT-2/DOPE vesicles or 1-3 microliters Lipofectamine 2000, diluted in 500 microliters of DMEM, was gently mixed (45 min) with 0.1 nmol ODNs, also diluted in 500 microliters of DMEM. The complexes were allowed to equilibrate for 20 min at room temperature, after which time they were immediately added to the cell cultures. After 6 h, the complexes were removed by washing with PBS. The cells were fixed with 4 % paraformaldehyde in PBS for 15 min, and permeabilized with 0.1 % Triton X-100 for 30 min. Subsequently, the cells were incubated with FluoroNanogoldstreptavidin for 2 h, washed three times with PBS, and immediately examined by epifluorescence microscopy. After examination, the cells were washed with 0.2 M sodium citrate, and enhanced with Nanoprobes HQ Silver . The cells were rinsed with PBS and postfixed with 1 % glutardialdehyde for 10 min. Finally, the samples were embedded in Epon, serially sectioned (60-nm thin sections), and examined at 60 kV.

Gold labeling was seen throughout the nucleoplasm, with little if any labeling in the nucleolus. Dense gold labeling was apparent on the ODN bodies, especially at their periphery. At higher magnification, gold labeling of ODNs in the nucleoplasm could be seen associated along the fibers of the nuclear matrix.

5HT1a autoreceptors expressed in RN46A cells, postsynaptic receptors expressed in SN48 cells, and receptors overexpressed in LLP-K1 cells were all efficiently downregulated following ODN delivery via a cationic lipid delivery system. These results demonstrate that the gene sequence per se, rather than intracellular factors, primarily determines the antisense effect. In addition, the delivery method is a relevant factor: antisense ODNs bind extensively to the RNA matrix in the cell nuclei, thereby interacting with target mRNA and causing its subsequent degradation. Antisense delivery effectively diminished the mRNA pool, thus resulting in downregulation of newly synthesized 5HT1A proteins, without the appearance of truncated protein fragments. In conjunction with the selected mRNA target sequences of the ODNs, the latter data indicated that effective degradation rather than steric blockage of mRNA impedes protein expression. The specificity of the antisense approach is reflected by the effective functional downregulation of the 5-HT1A receptor.

Reference:

Shi, F.; Visser, W. H.; de Jong, N. M.; Liem, R. S.; Ronken, E., and Hoekstra, D.: Antisense oligonucleotides reach mRNA targets via the RNA matrix: downregulation of the 5-HT1A receptor. Exp. Cell Res., 291, 313-325 (2003).

Abstract (Medline):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=14644154&dopt=Abstract

Nanoprobes has since introduced the brighter, more stable Alexa Fluor®* 488 and 594 FluoroNanogold in addition to the fluorescein form; these conjugates and their applications are described in full in our online catalog, linked below, and we plan to include other fluorophores in future. However, the preparation of probes, labels or tracers containing fluorescent and large gold labels is not feasible. Gold particles are good acceptors for fluorescence resonance energy transfer, or FRET, and absorb strongly at the emission wavelengths of commonly used fluorophores.

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).

Abstract (Medline):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9712158&dopt=Abstract

In order for fluorescence to be retained, the fluorescent tag has to be positioned sufficiently far away from the gold particle that a significant fraction is emitted directly rather than lost through energy transfer; generally, this means that the separation between the two must be greater than the Frster distance (the distance at which 50 % of absorbed energy is lost through energy transfer, and 50 % emitted as fluorescence). For Nanogold and fluorescein, the Frster distance is between 6 and 7 nm; FluoroNanogold-Fab' conjugates can fluoresce relatively strongly because Nanogold attached to one end and fluorescein at the other are further apart than this. However, for larger particles, such as 20 to 40 nm, the Frster distances are much larger, and commonly used biomolecular probes allow insufficient separation.

(Alexa Fluor is a trademark of Molecular Probes, Inc.)

More information:

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Combining Gold and Peroxidase Labeling for LM and EM In Neuroscience

We have previously described a number of articles in which immunogold labeling is combined with immunoperoxidase labeling for electron microscopy. Lujan and co-workers correlate this combination of techniques with light microscopy in their studies of the distribution of the voltage-gated potassium channel subunit Kv1.4 in the central nervous system of the rat and the contribution of this ion channel to neuronal functions.

A polyclonal antibody was raised in rabbits against the synthetic peptide corresponding to residues 13 - 37, near the putative N-terminus of the Kv1.4 gene product. For pre-embedding immunoperoxidase and gold labeling for EM, freefloating sections were incubated in 10% NGS diluted in TBS for 1 h, then incubated for 48 h in polyclonal antibody (code no. K8D) at a final protein concentration of 1 - 2 mg/ml diluted in TBS with 1% NGS. Immunoperoxidase labeling was conducted after several washes in TBS by incubation for 2 h in biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) diluted 1:50 in TBS / 1% NGS, followed by avidin-biotin-peroxidase complex (ABC kit, Vector Laboratories) diluted 1:100 for 2 h at room temperature. Peroxidase enzyme activity was revealed using 3,3' - diaminobenzidine tetrahydrochloride (DAB; 0.05% in TB, pH 7.4) as chromogen and 0.01% hydrogen peroxide as substrate. For immunogold labeling, sections were incubated in Nanogold® goat anti-rabbit IgG diluted 1:100 in TBS containing 1% NGS, washed several times in PBS, then postfixed in 1% glutaraldehyde in the same buffer for 10 min, washed in double distilled water, and enhanced using HQ Silver. Both peroxidase-reacted and the gold-silver labeled sections were then treated with 1% osmium tetroxide in 0.1 M phosphate), block-stained with uranyl acetate, dehydrated in graded series of ethanol and flat-embedded on glass slides in Durcupan (Fluka) resin. 70-90 nm thick sections from regions of interest were mounted on 200-mesh nickel grids and stained on drops of 1% aqueous uranyl acetate followed by Reynoldss lead citrate.

At the light microscopic level, the Kv1.4 subunit showed a unique distribution pattern, localized in specific neuronal populations of the rat brain. Regions expressing the highest levels of Kv1.4 protein included the cerebral cortex, the hippocampus, the posterolateral and posteromedial ventral thalamic nuclei, the dorsolateral and medial geniculate nuclei, the substantia nigra and the dorsal cochlear nucleus. The Kv1.4 subunit was mostly diffusely distributed; to a lesser extent, it stained cell bodies and proximal dendrites, and Kv1.4 immunoreactivity was also detected in nerve terminals and axonal terminal fields. At the electron microscopic level, Kv1.4 was located postsynaptically in dendritic spines and shafts at extrasynaptic sites, as well as presynaptically in axon and active zone of axon terminals, in the neocortex and hippocampus. Kv1.4 channels are shown to be widely distributed in the rat brain, and activation would have a variety of modulatory effects on neuronal excitability.

Reference:

Lujan, R.; de Cabo de la Vega, C.; Dominguez del Toro, E.; Ballesta, J. J.; Criado, M.; Juiz, J. M.; Immunohistochemical localization of the voltage-gated potassium channel subunit Kv1.4 in the central nervous system of the adult rat. J. Chem. Neuroanat., 26, 209-224 (2003).

Abstract (Medline):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=14615029&dopt=Abstract

More information:

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Nanogold® Labeling shows Relation of Proteins in Developing Rat Lens

Lo and co-workers have presented two studies in which pre-embedding Nanogold® and post-embedding gold labeling were used to study the relationships of different proteins in the developing rat lens, both of which use a well-defined three-step protocol. In the first, immunoelectron microscopy showed that the clathrin antibody was localized along the interlocking membrane, and branching actin filament networks were frequently observed within the cytoplasmic compartment of developing interlocking domains, consistent with the findings by fluorescence and immunogold labeling of the F-actin antibody in the domains; these results demonstrate for the first time that the clathrin/AP-2 complex plays a new role in the formation of interlocking domains in lens fiber cells. Branching actin networks and possibly other cytoskeletal components were found to be associated with the development and maintenance of these interlocking domains. In the second, immunogold TEM demonstrated that aquaporin 0 (formally MIP26) antibody was localized on membranous vesicles as well as plasma membranes of the cortical fiber cells, suggesting that a microtubule-based motor system exists in the lens and plays an important role in transporting membrane proteins such as aquaporin 0 in the vesicles during fiber cell differentiation and elongation.

For labelling with anti-clathrin and anti-MIP26 antibody, a three-step Nanogold pre-embedding procedure was used. Vibratome slices of formaldehyde-fixed rat lenses were fixed in 4% paraformaldehyde50 mM PBS (pH 73) for 50 min at room temperature (RT), washed in PBS overnight at 4°C, then cut into thick slices, and incubated in a blocking solution containing either 1% BSA and 03% Triton X-100 in PBS for 2 hr at room temperature. The tissue slices were incubated with primary antibody diluted in the blocking solution overnight at 4°C. After several washes in PBS, the lens slices were incubated in biotinylated goat anti-mouse IgG secondary antibody (1:100) in the blocking solution for 25 hr at RT, washed in PBS, followed by Nanogold-streptavidin (1:10) in 1% BSA, 005%TritonX-100 in PBS overnight at 4°C. The slices were washed in PBS, fixed with 25% glutaraldehyde in PBS for 30 minutes at RT, washed in PBS and water, enhanced with HQ Silver for 15 minutes at 18°C, rinsed in water and cacodylate buffer, pH 73, then fixed in 01% osmium tetroxide in 01 M cacodylate buffer, pH 73, for 30 min on ice and processed for TEM. Thin sections (80 nm) were stained with uranyl acetate and Reynolds lead citrate.

A three-step post-embedding procedure was used for labeling with anti-actin antibody: lenses were fixed immediately in 4% paraformaldehyde - 025% glutaraldehyde in 01 M cacodylate buffer (pH 73) for 1 hr at room temperature. Lenses were cut into 300 micrometer thick slices, dehydrated through a series of graded ethanol and embedded in LR White resin in gelatin capsules. Thin sections were cut and collected on nickel grids. The sections were blocked in a solution containing 5% dry milk and 2% BSA in PBS for 1 hr at RT, incubated on droplets of chicken anti F-actin polyclonal antibody(1:15 dilution), with 2% BSAPBS, pH 73 for 2 hr at room temperature. Grids were washed with PBS, incubated 1 hr on droplets of biotinylated goat anti-rabbit IgG (diluted 1:150) with 2% BSA-PBS, pH 73, washed with PBS, and finally incubated for 1 hr on droplets of streptavidin-10-nm gold diluted 1:25 with 2% BSAPBS, pH 73, and washed with PBS and distilled water. Grids were stained with uranyl acetate and Reynolds lead citrate.

References:

More information:

  • Nanogold conjugates - catalog information: www.nanoprobes.com/NanoAb.html
  • Nanogold conjugates - product info: www.nanoprobes.com/Inf2001.html
  • Colloidal gold - technical help: www.nanoprobes.com/TechCG.html
  • Nanogold Pre-embedding - References: www.nanoprobes.com/RefTopNG.html#Npre
  • Nanogold Post-embedding - References: www.nanoprobes.com/RefTopNG.html#Npost

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    Other Recent Publications

    Goode and co-workers describe the incorporation of the double labeling technique using silver-enhanced immunogold into a modified procedure that produces excellent labeling and ultrastructural preservation, even after exposure of ultrathin (90 nm) sections large enough to cover a 300-nm diameter singlehole grid to hot antigen retrieval solutions and prolonged labeling protocols.. Antigen retrieval was performed by immersion of grids bearing sections in 0.1 M sodium citrate buffer, pH 6.0, at 95°C for 10 min, cooling to room temperature for 20 min in the citrate buffer. Sections were then jet-washed (deionized water), immersed in drops of 0.5 M ammonium chloride in 0.1 M PBS, pH 7.3, for 20 min, then washed for 5 min in PBS, pH 7.3, containing 1% bovine serum albumin (BSA) and 0.1 % Tween-20 (washing buffer). Nonspecific binding was blocked using 20 % normal goat serum (NGS) diluted in washing buffer for 10 min. Sections were incubated overnight at 4°C in each of the primary antibodies, appropriately diluted in PBS, pH 7.3, containing 1% BSA, 0.1% Tween-20, and 5% NGS (antibody buffer). (in this case, alpha-Actinin, integrin beta-1, and synaptopodin supernatant antibodies, respectively diluted 1:50; 1:10, and 1:2). After rinsing in three changes of washing buffer for 5 min each, sections were incubated in blocking buffer for 20 min at 20°C, followed by a 2-hr incubation at 20°C in goat anti-mouse IgG conjugated to 10-nm gold particles diluted 1:100 in antibody buffer. Sections were jet-washed, silver-enhanced for 12 min according to Holgate's procedure (1986), then washed again in deionized water for 2 min before the silver stain was fixed for 2 min in 3 % sodium thiosulfate. Before application of the second monoclonal antibody, sections were jet-washed; the procedure was repeated as for the first primary antibody. The sections were silver-enhanced for 6 min, jet-washed again, and counterstained in saturated aqueous uranyl acetate for 3 min before being finally rinsed briefly in distilled water and allowed to dry.

    Reference:

    Goode N. P.; Shires M.; Crellin D. M.; Khan T. N., and Mooney A. F.: Post-embedding Double-labeling of Antigen-retrieved Ultrathin Sections Using a Silver Enhancement-controlled Sequential Immunogold (SECSI) Technique. J. Histochem. Cytochem., 52, 141-144 (2004).

    Abstract (courtesy of the Journal of Histochemistry and Cytochemistry):
    http://www.jhc.org/cgi/content/abstract/52/1/141

    A novel method for preserving the fine structure of adult Caenorhabditis elegans tissues well enough to take advantage of the nanometer-scale resolution of immunogold labeling is described in the current Journal of Histochemistry and Cytochemistry. The animals cuticle slows the diffusion of solutions into the animal and thus makes it difficult to preserve both immunoreactivity and cell morphology. Rostaing and co-workers, however, instantly immobilized tissue in vitreous ice by freezing living adult animals under high pressure. Frozen specimens were then chemically fixed, dehydrated, and embedded at low temperatures. As a result, chemical diffusion across the cuticle could occur over an extended period without morphological deterioration.

    Reference:

    Rostaing, P.; Weimer, R. M.; Jorgensen, E. M.; Triller, A.; and Bessereau, J. L.: Preservation of Immunoreactivity and Fine Structure of Adult C. elegans Tissues Using High-pressure Freezing. J. Histochem. Cytochem., 52, 1-12 (2004).

    Abstract (courtesy of the Journal of Histochemistry and Cytochemistry):
    http://www.jhc.org/cgi/content/abstract/52/1/1

    Esumi and co-workers have investigate the use of composites of 5.6 - 7.5 nm silver, 1.2 - 1.6 nm platinum and 1.6 - 2.0 nm palladium nanoparticles with polyamidoamine (PAMAM) and poly(propyleneimine) (PPI) dendrimers (generations 2, 3, and 4) with surface amino groups, prepared in aqueous solution, as catalysts for the reduction of 4-nitrophenol. The rate constants for the reduction are very similar between PAMAM and PPI dendrimer -silver nanocomposites, whereas the rate constants for the PPI dendrimer-platinum and -palladium nanocomposites are greater than those for the corresponding PAMAM dendrimer nanocomposites, possibly because the PPI dendrimers, being more hydrophobic, are less strongly adsorbed to the metal nanoparticles thus allowing greater surface reactivity. In addition, it is found that the rate constants for the reduction of 4-nitrophenol involving all the dendrimer-metal nanocomposites decrease with an increase in the dendrimer concentrations, and the catalytic activity of dendrimer-palladium nanocomposites is highest.

    Reference:

    Esumi, K.; Isono, R., and Yoshimura, T.: Preparation of PAMAM- and PPI-Metal (Silver, Platinum, and Palladium) Nanocomposites and Their Catalytic Activities for Reduction of 4-Nitrophenol. Langmuir, 20, 237-243 (2004).

    Article information (courtesy of the American Chemical Society):
    http://dx.doi.org/10.1021/la035440t

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