The small size of Nanogold® probes and their consequent ability to access nuclear antigens, combined with the high sensitivity obtained with silver enhancement, makes them excellent reagents for ultrasensitive in situ hybridization detection of specific DNA sequences, both with and without in situ PCR.

Striking results have been obtained with amplification of the target signal using biotinylated tyramide (Catalyzed Reporter Deposition or CARD, also known as Tyramide Signal Amplification or TSA): detection of the product with Nanogold®-streptavidin and silver enhancement results in clear signals even in cells with only one or two copies of the target gene (see: Hacker, G. W., et al.: Cell Vision, 4, 54-65 (1997)). Similar sensitivity is seen in RNA detection (see: Zehbe, I., et al.: Am. J. Pathol., 150, 1553-1561 (1997)).

Advantages

• More sensitive than NBT-BCIP alkaline phosphatase or peroxidase in combination with label amplification for single gene copy resolution.
• No need for more lengthy PCR procedures for most cases: avoids false positives due to mispriming and amplicon diffusion.
• Black color easily seen with standard brightfield microscope. No expensive fluorescent optics required, and no problems with autofluorescence or bleaching.
• Black signal color is compatible with full strength standard cell and nuclear stains (H & E, nuclear fast red, methyl green).
• High spatial resolution signal which may be used for EM studies as well.

Single copies of HPV 16 in SiHa cells detected by CARD-amplified Nanogold®-silver ISH. This cell line derived from cervical carcinoma contains only one to a few copies of HPV 16, which appear as single spots. Cytospin preparation, counterstained with Nuclear Fast Red (light micrograph courtesy of Dr. G. Hacker, Landeskrankenstalten, Salzbrg. Austria).

Nanogold® sets an unrivaled performance standard for electron microscopy.

Nanogold® conjugates diffuse easily tens of microns into tissues, permeabilized cells and nuclei, where most colloidal gold probes have difficulty. Staining is more stoichiometric, with lower backgrounds.

Although the 1.4 nm Nanogold® particles are visible directly at 50-120 kX magnification even in 80 nm sections (in otherwise unstained samples) they are more easily visualized after a brief (1-5 min) enhancement with silver developer (LI Silver or HQ Silver). The resulting 2 to 100 nm silver grains (size depends on development time) are easily seen at lower magnification even with uranyl acetate, osmium, or lead citrate staining.

This scanning transmission electron microscope (STEM) image clearly shows that labeling with Monomaleimido-Nanogold® (arrows) occurs specifically at a hinge thiol site on the IgG molecule.

• Nanogold® probes have excellent penetration into permeabilized cells and nuclei, similar to immunofluorescent probes and far better than colloidal gold probes.
• Nanogold®-Fab’ probes are smaller than immunofluorescent IgG labels.
• There is absolutely no aggregation with Nanogold® probes, and they are very stable since the Nanogold® is covalently linked to the conjugate biomolecule.
• With a subsequent brief (5-15 min) silver enhancement (LI Silver) they give a signal visible even in ordinary brightfield optical microscopes (UV optics are not necessary) with a sensitivity that is usually better than fluorescence.
• Since the same Nanogold® probe can be used for the EM as well, preliminary staining can be followed in the light or confocal microscope while a parallel sample can be examined at the ultrastructural EM level.
• This flexibility is not available with fluorescent probes. Nanogold® is now available conjugated to streptavidin and anti-biotin Fab’ for nucleic acid detection.

Spindle microtubules labeled with anti-tubulin primary antibody followed by (LEFT) goat anti-mouse colloidal gold or (RIGHT) goat Fab’ anti-mouse-Nanogold (Light micrograph courtesy of Dr. D. Vandré and Dr. R. Burry, Ohio State University. Original magnification = 1300x).

Nanogold®-antibody conjugates have been used to detect < 0.1 pg (7 X 10-19 moles) of antigen on immunodot blot assays. This sensitivity is better than most radioactive, enzymatic, colloidal gold, or fluorescence techniques. In fact, Nanogold® labeling is one of the most sensitive assays known. It rivals chemiluminescence, but provides a permanent record without the nuisance of film, development and darkroom procedures, and in a much shorter time.

Nanogold® reagents may also be used for Westerns, and our Nanogold®-streptavidin or Nanogold®-anti-biotin conjugates provide ultrasensitive nucleic acid detection.

Nanogold®-anti-mouse Fab’ blotted against mouse IgG, developed with LI Silver (Nanoprobes). These ultra small gold particles nucleate silver deposition so well that unprecedented sensitivity is achieved. This immunodot blot shows 0.1 pg sensitivity (arrow).

After a protein or other molecule is labeled with Nanogold®, it may be run on a polyacrylamide gel. Subsequently, it may be developed with LI Silver or GoldEnhance™ to specifically stain the gold-containing bands.

The process is rapid, sensitive and selective, only taking a few minutes for dense staining to develop. It also works on transfer blots. This may be used to distinguish which components are labeled with Nanogold®