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In pathology, silver staining is the use of silver to selectively alter the appearance of a target in light microscopy of histological sections; in temperature gradient gel electrophoresis; and in polyacrylamide gels.
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 . 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 color 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.
Silver nitrate forms insoluble silver phosphate with phosphate ions; this method is known as the Von Kossa Stain. When subjected to a reducing agent, usually hydroquinone, it forms black elementary silver. This is used for study of formation of calcium phosphate particles during bone growth.
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.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. 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.
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