In Situ Hybridization with Nanogold®-Streptavidin
Diagram 1: Schematic representation of the Nanogold-Silver In Situ Hybridization technique. The DNA sequence in the host cell is detected by hybridization with a biotin-labeled cDNA probe. Biotin bound covalently with spacer arms is then detected using streptavidin-Nanogold and subsequently silver-enhanced using silver acetate or silver lactate autometallography. For simplicity, only four such biotin and streptavidin-Nanogold molecules are shown.
Hacker, G. W., Hauser-Kronberger, C., Zehbe, I., Su, H., Schiechl, A., Dietze, O., and Tubbs, R.; Cell Vision, 4, 54-65 (1997).
© Eaton Publishing Co.; used by permission.
Detection of the Human Papillomavirus type 6/11 genome using this method is shown below; as can be seen, the Nanogold®-silver staining gives a dense black signal which in many cases is clearer than that obtained using conventional enzyme substrates.
HPV 6/11 in condyloma acuminatum detected by Nanogold-silver ISH. In this case, strong labeling was obtained. Most infected nuclei showed dense black or granulated black staining. A few nuclei also showed single black spots of labeling, indicating the presence of very low copy numbers of this DNA virus. Staining was was as described in the protocol; formalin-fixed, 7-micrometer-thick paraffin section, counterstained with hematoxylin and eosin. Magnification, X 285.
Diagram 2: Schematic representation of the CARD-Nanogold-Silver In Situ Hybridization technique. The RNA sequence in the host cell is detected by hybridization with a FITC-labeled riboprobes. A first antibody layer directed against FITC then binds to the FITC molecule used as the nucleic acid reporter molecule. The next layer is a secondary antibody against the IgG of the species prviding the primary antibody, labeled with biotin. For simplicity, only one of each of these two antibodies is shown. Biotin is then bound to Streptavidin-biotin-peroxidase (S-ABC-Peroxidase) complexes. Four such molecules are shown, each of them carrying peroxidase. These peroxidase molecules are then catalyzing the accummulation of biotinylated tyramide (catalyzed reporter deposition), which finally leads to a "multiplication" of the biotin label. As a final layer, streptavidin-Nanogold with subsequent silver acetate or silver lactate amplification (autometallography) is used. Each of these final streptavidin molecules can detect one of many molecules of biotin. In this way, an enormous amplification is reached, in many cases leading to single gene copy sensitivity without the necessity of PCR or 3SR techniques.
Hacker, G. W., Hauser-Kronberger, C., Zehbe, I., Su, H., Schiechl, A., Dietze, O., and Tubbs, R.; Cell Vision, 4, 54-65 (1997).
© Eaton Publishing Co.; used by permission.
This procedure was used for the detection of HPV-16, a cDNA virus, in SiHa cells cultured from cevical cancer specimens. These cells are known to contain only one to a few copies of the viral DNA; Nanogold®-streptavidin in situ hybridization in conjunction with CARD gave dense black granular staining of the cell nuclei; the black signal was more readily distinguished than the signal produced by peroxidase-streptavidin. The procedure may be completed in 5 to 7 hours;
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 by the technique applied (see protocol for details). Cytospin preparation, counterstained with Nuclear Fast Red. Similar staining is obtained in tissue sections of formalin-fixed paraffin material (Zehbe, et al, Am. J. Pathol. 150, 1553-1561 (1997)). Original magnification, X 560.
Epstein-Barr virus in Hodgkins Disease; an example of highly sensitive detection using the RNA detection protocol. Original magnification, X 400.
Ki-67 breast carcinoma antigen detected using indirect Nanogold® labeling. Ki-67 antigen was detected using a primary monoclonal antibody followed by a biotinylated goat anti-mouse secondary; this was visualized using Nanogold®-streptavidin (250-fold dilution) followed by autometallography. Original magnification, X 400.
2016 NANOGOLD®- streptavidin (NS)
1.4 nm gold particle is attached to streptavidin.
Instructions for use of Nanogold®-Streptavidin
A complete kit for In Situ detection using Nanogold®-Streptavidin is under development.
For more information contact:
Other Applications: