Differential staining of DNA strand breaks in dried comet assay slides

The comet assay involves embedding cells in agarose on microscope slides. A fter lysis and electrophoresis, staining is usually performed with a fluore scent DNA-binding dye and observation is carried out on fresh wet slides th rough an epifluorescence microscope. We present here a simple alternative f or preservation of the agarose comet slides and a fluorescent staining that allows fine differential analysis of DNA strand breaks under confocal micr oscopy. Lymphocytes were processed according to previous published methods. Slides were quickly dehydrated in a hot oven at 50C for 20 min. Once the a garose layer was dried and reduced to a thin film, slides were treated with RNase. image analysis showed higher tail length, total area, and tail mome nt. Using confocal microscopic optical sectioning, a thickness of approxima tely 180 mum for wet slides and 12 mum for dehydrated gels was calculated. Acridine orange, used for DNA differential staining, allowed quantitation o f metachromasia and orthochromasia with confocal scanning microscopy. Diffe rences between alkaline and neutral comet assay with AO were clear-cut and, in principle, a metachromatic index can be calculated.