Silver enhancement of nanogold and undecagold

A freeze substitution fixation-based gold enlarging technique for EM studies of endocytosed Nanogold-labeled molecules

“…Although the small size of 1.4 nm Nanogold makes it ideal for preserving the proper binding and function of a labeled ligand, these clusters were not large enough to be located directly in EM projection images and in most tomograms derived from stained cell sections. We therefore adapted autometallography and nanoparticle seeding techniques that are used to enlarge 1.4 nm Nanogold clusters by selectively depositing silver and/or gold atoms onto their surfaces (Busbee et al, 2003;Daniel and Astruc, 2004;Gole and Murphy, 2004;Hainfeld and Furuya, 1995;Hainfeld et al, 1999;Jana et al, 2001;Meltzer et al, 2001;Okitsu et al, 2005;Zou et al, 2006).…”

“…The FSF procedure described above was modified to include a preembedding gold-enlarging technique for HPF cells by adapting approaches that involve silver or gold enhancement at room temperature (Danscher, 1981;Hacker et al, 1988;Hainfeld and Furuya, 1995;Scopsi, 1989), gold-toning (Sawada and Esaki, 2000), seed-mediated gold-enlarging (Busbee et al, 2003;Daniel and Astruc, 2004;Gole and Murphy, 2004;Handley, 1989;Jana et al, 2001;Meltzer et al, 2001;Okitsu et al, 2005;Zou et al, 2006) and a FSF-based silver-enhancement procedure (Morphew et al, 2007). To avoid the background that results from spontaneous autonucleation, we designed a three-step enlarging protocol in which silver enhancement was used to slightly enlarge the Nanogold, the silver shell was coated by gold toning to make it insoluble in osmium, and the particles were further enlarged to 10 -16 nm using gold enhancement.…”

“…However, Nanogold is too small to see by EM in most cellular samples, so it must be enlarged after it has been internalized and the cells have been fixed. Methods to enhance Nanogold by deposition of silver or gold atoms on clusters in chemically-fixed samples are available (Danscher, 1981;Hacker et al, 1988;Hainfeld and Furuya, 1995;Scopsi, 1989), but when we applied them to chemically-fixed tissue samples containing an endocytosed 1.4 nm Nanogold-ligand, they resulted in high backgrounds due to auto-nucleation and none of these methods is compatible with HPF/FSF samples. Thus, there is a need for methods for enhancing small gold particles after either HPF/FSF or an optimized chemical fixation and before cells are embedded in plastic for microtomy.…”

New Frontiers in Gold Labeling

“…Conjugates are small enough to run on gels. The gold retards proteins by the approximate weight of the gold ( ‫ف‬ 5 kD for undecagold and ‫ف‬ 15 kD for Nanogold) (Weinstein et al 1989;Hainfeld and Furuya 1995). One can then stain with Coomasssie and, in parallel with silver, track the labeling on a molecular basis (Gregori et al 1997).…”

“…However, gold clusters (undecagold ‫5ف‬ kD and Nanogold ‫51ف‬ kD) attached to proteins shift the weight by approximately that amount. This means that Coomassie blue staining can be used to visualize proteins, and silver staining will reveal the gold-labeled bands (using a parallel gel, or a lane cut lengthwise, half for Coomassie, half for silver) (Hainfeld and Furuya, 1995;Gregori et al 1997).…”