| ||Silver Enhancement of Colloidal Gold |
Silver enhancement protocols [from: Stierhof et al. (1992 & 1995)]
This protocol is taken from the 1999 FEBS course manual and was contributed by Heinz Schwarz.
|General remarks: |
Labeled sections have to be fixed to avoid loss of label during the enhancement procedure and thoroughly washed with bi-distilled water to remove ions (especially chloride ions) which could interfere with the silver-enhancement process. Enhancement is carried out by incubating the grids on a drop of ~100 µl of the enhancer solution. Although most of the enhancers can even be used in daylight, we recommend covering the grids or working under red safe light conditions, as this delays self-nucleation of silver ions. The reaction is stopped by transfering the grids to bi-distilled water. Before staining with uranyl acetate and lead citrate, components like gum arabic have to be completely removed by thoroughly washing the grids. It has to be noted that higher temperatures and moving of the grids during incubation on the enhancer solution speed up the deposition of silver on the gold surface.
- Silver lactate, acidic (Danscher, 1981)
- 0.6 ml gum arabic (33% in bidistilled water) plus 0.1 ml citrate buffer (2.55 g citric acid plus 2.35 g trisodium citrate dihydrate, add bi-distilled water to make 10 ml, pH 3.8) plus 0.15 ml hydroquinone (0.85 g in 15 ml bidistilled water) plus 0.15 ml silver lactate (0.11 g in 15 ml bi-distilled water). Gum arabic, citrate buffer, and hydroquinone can be premixed and stored in a freezer. Silver lactate has to be stored separately in a freezer. Incubation time for 1-nm gold is about 20 min, for Nanogold about 25 min (20 - 22°C).
- Silver lactate (Lah et al., 1990)
- As above, replace citrate buffer by 0.2 M HEPES buffer, pH 6.8. Incubation time ~3 min. for 1-nm gold, for Nanogold 4 to 5 min (20 - 22°C). The final pH is 4 (see Burry 1995, p. 220). Remove blocking buffer, add 25 µl of the primary antibody solution per coverslip (with a final concentration in the range of 1-5 µg specific IgG/ml) and incubate for 30 -60 min.
- Silver acetate, acidic (Hacker et al., 1988)
- Mix equal amounts of 0.5% hydroquinone in 0.5 M citrate buffer (see above), and 0.2% silver acetate. Final concentration of 16.5% gum arabic was prepared from a stock solution of 33% gum arabic. Incubation time ~90 min for 1-nm gold (20 - 22°C) (but only ~20 min without gum arabic).
- HQ SILVERª.(Nanoprobes)
- Equal amounts of the three components initiator, moderator, and activator were mixed before use (see instructions of Nanoprobes). Incubation time ~3 min for 1-nm gold and ~5.5 min for Nanogold (20 to 22°C). HQ SILVER contains a protective colloid.
Most of the commercially available enhancers like IntenSE M (Amersham), R-Gent (Aurion) and SEKL15 (British BioCell) appear to be less efficient. They can be improved by adding the protective colloid gum arabic (for details see Stierhof et al 1991, 1992, 1995).
- Danscher G (1981) Histochemistry 71, 1-16..
- Lah JJ, Hayes DM, and Burry RW (1990) J Histochem Cytochem 38, 503-508.
- Burry RW (1995) In: Immunogold-Silver Staining. Principles, Methods, and Applications. Ed. M. A. Hayat, CRC Press, Boca Raton, pp. 217-230.
- Hacker GW, Grimelius L, Danscher G, Bernatzky G, Muss W, Adam H, and Thurner J (1988) J Histotechnol 11, 213-221.
- Stierhof Y-D, Humbel BM, and Schwarz H (1991) J Electron Microsc Tech 17, 336-343.
- Stierhof Y-D, Humbel BM, Hermann R, Otten MT, and Schwarz H (1992) Scanning Microscopy 6, 1009-1022.
- Stierhof Y-D, Hermann R, Humbel BM, and Schwarz H (1995) In: Immunogold-Silver Staining. Principles, Methods, and Applications. Ed. M. A. Hayat, CRC Press, Boca Raton, pp. 97-118.
- see related articles in: Immunogold-Silver Staining. Principles, Methods, and Applications. Ed. MA Hayat, CRC Press, Boca Raton (1995). and in: Colloidal Gold: Principles, Methods, and Applications. Vol 1. Ed. MA Hayat, Academic Press, San Diego (1989).