Immunocytochemical Methods and Protocols

Volume 34 of the series Methods in Molecular Bilogy pp 211-216

Use of Immunogold with Silver Enhancement

  • Constance OliverAffiliated withOffice of Naval Research

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Although gold particles are readily detectable by transmission electron microscopy, they can be difficult to visualize by bright-field light microscopy. If the particle size is large enough and the labeling dense enough, the gold particles will stain tissue red (1, 2). However, unless the gold is silver-enhanced to help visualize it, the sensitivity of the staining is fairly low. During silver-enhancement, the colloidal gold serves as a nucleation site for the deposition of metallic silver. The silver layer increases the size of the gold and imparts a black color to the stained tissue when viewed by bright-field microscopy (Fig. 1A). The silverenhanced gold particles can also be visualized using epipolarization, where they appear bright against a dark background (Fig. 1B). The silver enhancement method has its basis in 19th-century photographic techniques. The enhancing solutions are physical developers that contain both silver ions and a reducing agent, buffered to an acid pH. The developers most commonly used contain silver lactate as the source of silver ions (3). The silver lactate has a low dissociation coefficient that allows for more control of the reduction. Hydroquinone ( 1,4-dihydroxybenzene) is the only reducing agent that has been used in silver-enhancement techniques. A protective colloid, such as gum arabic, bovine serum albumin, dextran, polyethylene glycol (PEG), or polyvinylpyrrolidone (PVP), is frequently added in order to inhibit the autocatalytic reaction between the silver salt and the reducing agent.