Background Information

Simultaneous staining of DNA with the intercalating dye PI and detection of incorporated BrdU by antibody requires the presence of both double-stranded DNA (to be stainable with PI) and denatured DNA (to have incorporated BrdU accessible to antibody). A variety of methods and modifications have been proposed to ensure that the degree of DNA denaturation is optimal for simultaneous staining with PI and detection of BrdU (for reviews see Dolbeare and Selden, 1994, and Gray et al., 1990). Alternatives to heat- or acid-induced DNA denatu-ration as presented in this unit include partial DNA cleavage with exonuclease III or EcoRI, which expose BrdU to the antibody and still preserve sections of DNA in double-stranded conformation, reactive with PI (Dolbeare and Gray, 1988). This procedure, therefore, although more complex and expensive, can be used in situations when it is necessary to preserve (e.g., for immunocytochemical detection) other antigens that are degraded, extracted, or denatured by heat or acid treatment.

Cytochemical methods of BrdU detection (Latt, 1973, 1977; Darzynkiewicz et al., 1978) are not discussed here. These methods are reviewed by Crissman and Steinkamp (1990). Several of these techniques, including simultaneous differential detection of BrdU and iodo-deoxyuridine (IdU), are presented elsewhere (Darzynkiewicz et al., 1994).

Still another approach for BrdU detection is based on selective photolysis of DNA containing the incorporated BrdU. The photolytically generated DNA strand breaks are subsequently labeled with fluoresceinated nucleotides in a reaction catalyzed by exogenous terminal transferase, referred to as DNA strand break induction by photolysis (SBIP; Li and Dar-

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