Immunogold staining

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Immune gold staining of the virus PMMoV (pictures c and d)

The immunogold staining (Engl. Immunogold staining ) is an immunostaining , as reporter signal colloidal gold wear particles. It is used in sample preparation for transmission electron microscopy (TEM).

principle

The immunogold staining is based on the immunostaining for marking individual antigens , however gold particles with a diameter of 5, 10 or 20 nm are coupled to the secondary antibody . With the different diameters, different antigens can be marked in parallel. The gold particles have a comparatively high electron density and are therefore less permeable to the cathode ray , which means that the particles appear as dark, high-contrast points in the TEM image.

To prepare samples of fixed tissues , thin sections are made with a microtome . In order to reduce aldehydes in the preparation, the preparation is reduced in 0.1% (m / V) sodium borohydride for 5 minutes , followed by several washes with PBS or TBS buffer . Suspensions of viruses , on the other hand, are sedimented with an airfuge on a PVA -coated copper grid with 100,000 g. Analogously to the immunostaining, the free protein binding sites in the preparation are then blocked for 20 minutes with a blocking solution (5% bovine serum albumin in PBS or TBS). The first antibody (primary antibody) is bound in a PBS- or TBS-based solution with 1% bovine serum albumin. After further washing steps in TBS or PBS with 0.1% bovine serum albumin, incubation with the gold-marked secondary antibody in PBS or TBS follows. Lower concentrations of antibodies with longer incubation times produce more specific staining. Alternatively, a gold-labeled protein A or protein G can be used instead of the gold-labeled secondary antibody . A subsequent silver coloration lowers the detection limit by a factor of 200. This means that the marking can also be observed with a light microscope .

Immunogold staining is also used for scanning tunneling microscopy .

history

Immunogold staining was published in 1971 by WP Faulk and GM Taylor.

literature

  • J. Roth: The silver anniversary of gold: 25 years of the colloidal gold marker system for immunocytochemistry and histochemistry. In: Histochemistry and cell biology. Volume 106, Number 1, July 1996, pp. 1-8, PMID 8858362 .
  • F. D'Amico, E. Skarmoutsou: Quantifying immunogold labeling in transmission electron microscopy. In: Journal of microscopy. Volume 230, Pt 1April 2008, pp. 9-15, doi : 10.1111 / j.1365-2818.2008.01949.x , PMID 18387034 .
  • MS Sirerol-Piquer, A. Cebrián-Silla, C. Alfaro-Cervelló, U. Gomez-Pinedo, M. Soriano-Navarro, JM Verdugo: GFP immunogold staining, from light to electron microscopy, in mammalian cells. In: Micron. Volume 43, Number 5, April 2012, pp. 589-599, doi : 10.1016 / j.micron.2011.10.008 , PMID 22227011 .

Individual evidence

  1. ^ J. Roth, M. Binder: Coloidal gold, ferritin and peroxidase as markers for electron microscopic double labeling lectin techniques. In: The journal of histochemistry and cytochemistry: official journal of the Histochemistry Society. Volume 26, Number 3, March 1978, pp. 163-169, doi : 10.1177 / 26.3.632554 , PMID 632554 .
  2. ↑ J. Roth, M. Bendayan, L. Orci: Ultra Structural localization of intracellular antigens by the use of protein A-gold complex. In: The journal of histochemistry and cytochemistry: official journal of the Histochemistry Society. Volume 26, Number 12, December 1978, pp. 1074-1081, doi : 10.1177 / 26.12.366014 , PMID 366014 .
  3. ^ MA Hayat: Immunogold-Silver Staining. CRC Press, 1995, ISBN 978-0-849-32449-9 .
  4. ↑ O. Van Laere, L. De Wael, J. De Mey: Immuno gold staining (IGS) and immuno gold silver staining (IGSS) for the identification of the plant pathogenic bacterium Erwinia amylovora (Burrill) Winslow et al. In: Histochemistry. Volume 83, Number 5, 1985, pp. 397-399, PMID 2416717 .
  5. ↑ C. Oliver: Use of immunogold with silver enhancement. In: Methods in molecular biology. Volume 115, 1999, pp. 241-245, doi : 10.1385 / 1-59259-213-9: 241 , PMID 10098186 .
  6. ^ R. Hermann, P. Walther, M. Müller: Immunogold labeling in scanning electron microscopy. In: Histochemistry and cell biology. Volume 106, Number 1, July 1996, pp. 31-39, PMID 8858365 .
  7. ^ WP Faulk, GM Taylor: An immunocolloid method for the electron microscope. In: Immunochemistry. Volume 8, Number 11, November 1971, pp. 1081-1083, PMID 4110101 .