In traditional stained glass, silver stain is a technique to produce yellow to orange or brown shades (or green on a blue glass base), by adding a mixture containing silver compounds (notably silver nitrate), and firing lightly. It was introduced soon after 1800, and is the "stain" in the term "stained glass". Silver compounds are mixed with binding substances, applied to the surface of glass, and then fired in a furnace or kiln.
Silver staining was introduced by Kerenyi and Gallyas as a sensitive procedure to detect trace amounts of proteins in gels. The technique has been extended to the study of other biological macromolecules that have been separated in a variety of supports.
Classical Coomassie Brilliant Blue staining can usually detect a 50 ng protein band; silver staining increases the sensitivity typically 50 times.
Many variables can influence the colour intensity and every protein has its own staining characteristics; clean glassware, pure reagents, and water of highest purity are the key points to successful staining.
Some cells are argentaffin. These reduce silver solution to metallic silver after formalinfixation. Other cells are argyrophilic. These reduce silver solution to metallic silver after being exposed to the stain that contains a reductant, for example hydroquinone or formalin.
Silver staining aids the visualization of targets of interest, namely intracellular and extracellular cellular components such as DNA and proteins, such as type III collagen and reticulin fibres by the deposition of metallic silver particles on the targets of interest.
Silver staining is used in karyotyping. Silver nitrate stains the nucleolar organization region (NOR)-associated protein, producing a dark region wherein the silver is deposited and denoting the activity of rRNA genes within the NOR. Human chromosomes 13, 14, 15, 21, and 22 have NORs, which increase the silver stain activity by at least 50 times.
First, the proteins are denatured in the gel by a fixative solution of 10% acetic acid and 30% ethanol and precipitated, at the same time the detergent (mostly SDS) is extracted. The diffusion of the proteins is thus significantly reduced. After repeated washing with water, the gel is incubated in a silver nitrate solution. Silver ions bind to negatively charged side chains of the proteins. Excess silver ions are then washed off with water. In the final development step, the silver ions are reduced to elemental silver by addition of alkaline formaldehyde. This stains the sites where proteins are present, brown to black.
The intensity of the staining depends on the primary structure of the protein. Furthermore, the cleanliness of the vessels used and the purity of the reagents influence the silver stain. Common artifacts in silver stained gels are bands of keratin in the ranges of 54-57 kDa and 65-68 kDa as a contamination of the sample prior to the electrophoresis.
Methenamine silver stains
There are several silver stains incorporating methenamine, including:
A silver stain (GMS) demonstrating the fungus Histoplasma (black round balls) in a liver biopsy.
A number of DNA samples from specimens of Littorina plena amplified using PCR with primers targeting a variable simple sequence repeat (SSR, a.k.a. microsatellite) locus. Samples have been run on a 5% polyacrylamide gel and visualized using silver staining.
^Kerenyi L, Gallyas F (1973). "Über Probleme der quantitiven Auswertung der mit physikalischer Entwicklung versilberten Agarelektrophoretogramme". Clin. Chim. Acta. 47 (3): 425-436. doi:10.1016/0009-8981(73)90276-3. PMID4744834.
^Switzer RC 3rd, Merril CR, Shifrin S (Sep 1979). "A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels". Anal. Biochem. 98 (1): 231-237. doi:10.1016/0003-2697(79)90732-2. PMID94518.CS1 maint: multiple names: authors list (link)
^Hempelmann E, Schulze M, Götze O (1984). "Free SH-groups are important for the polychromatic staining of proteins with silver nitrate". Neuhof V (Ed)Electrophoresis '84, Verlag Chemie Weinheim 1984: 328-330.
^Schwint OA, Labraga M, Cervino CO, Haffar M, Sequeiros PH, Marcos HJ (2004). "A modification of the staining technique of reticular fibres for image analysis of the cardiac collagen network". Cardiovasc. Pathol. 13 (4): 213-20. doi:10.1016/S1054-8807(03)00153-4. PMID15210137.
^R. C. Switzer, C. R. Merril, S. Shifrin (September 1979), "A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels", Anal Biochem (in German), 98 (1), pp. 231-237, doi:10.1016/0003-2697(79)90732-2, PMID94518CS1 maint: multiple names: authors list (link)
^Rabilloud T, et al. (1988). "Improvement and simplification of low-background silver staining of proteins by using sodium dithionite". Electrophoresis. 9 (6): 288-291. doi:10.1002/elps.1150090608. PMID2466660.
^E. Hempelmann, M. Schulze, O. Götze: Free SH-groups are important for the polychromatic staining of proteins with silver nitrate. In: V. Neuhof (Editor): Electrophoresis. Verlag Chemie, Weinheim, 1984, pp. 328-330.
 Hempelmann E. SDS-Protein PAGE and protein detection by silverstaining and immunoblotting of Plasmodium falciparum proteins. in: Moll K, Ljungström J, Perlmann H, Scherf A, Wahlgren M (eds) Methods in Malaria Research, 5th edition, 2008, 263-266