The single-cell gel assay (comet assay) is a very useful microelectrophoretic technique for evaluation of DNA damage and repair in individual cells. Usually, the comets are visualized and evaluated with fluorescent DNA stains. This staining requires specific equipment (e.g., a high-quality fluorescence microscope), the slides must be analyzed immediately, and they cannot be stored for long periods of time. Here we describe, using human lymphocytes, some modifications of the silver staining for comets that significantly increase the sensitivity/reproducibility of the assay. This silver staining was compared with fluorescence staining and commercial silver stains

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Journal of Histochemistry and Cytochemistry, Vol. 49, 1183-1186, September 2001

 After electrophoresis, the agarose gels containing the cells were placed on microscope slides and washed twice for 2 min with deionized water. The gels were dried 1 hr at room temperature (RT) and fixed for 10 min in a solution containing 15% w/v trichloroacetic acid, 5% w/v zinc sulfate, and 5% glycerol. After fixation the slides were washed three times in deionized water and dried for approximately 5 hr at RT. Before silver staining, the gels were re-hydrated for 5 min in deionized water. The staining solution was prepared fresh before use and added to the samples very gently. The solution was prepared in the following sequence: 34 ml of Solution B (0.2% w/v ammonium nitrate, 0.2% w/v silver nitrate, 0.5% w/v tungstosilicic acid, 0.15% v/v formaldehyde, and 5% w/v sodium carbonate) to 66 ml of Solution A (5% sodium carbonate). The slides were immersed for 20 min (approximately the time required to obtain a light gray color) and placed in a shaker in small glass boxes covered with aluminum foil. After staining, the slides were washed three or four times in deionized water. The staining was stopped by immersing the slides for 5 min in 1% acetic acid solution, followed by two washes in deionized water, and the slides were air-dried. All the glass materials used for the silver staining were pretreated with 50% of nitric acid and then washed with detergents and several times with deionized water. The assay was performed in duplicate and we tested our method of silver staining in parallel with (a) Trevigen silver staining, (b) Bio-Rad Silver Stain Plus Kit (Hercules, CA), and (c) fluorescence (propidium iodide). For Trevigen we applied the protocol supplied in Trevigen, Inc. (1999) and for Bio-Rad silver staining we followed the manufacturer's instructions. For the propidium iodide staining, after electrophoresis the gels were rinsed in ultra-pure water and stained with 2.5 μg/ml of propidium iodide dissolved in 0.1 M NaCl for 15 min. The slides were viewed and photographed using a fluorescence microscope (Nikon Opti Phot-2 UFX-IIA)