Indeed, the acid- or heat- induced denaturation of DNA to make BrdU accessible to antiBrdU mAb is incompatible with immunocytochemical detection of many proteins including cell surface markers. DNase tretament, on the other hand, is hard to control and is often associated with such loss of DNA that it is difficullt to identify the cell cycle position or DNA ploidy. The SPIB (Strand Break Induction by Photolysis) methodology has no such limitations. The cells are UV-illuminated to selectively photolyze DNA in the cells that incorporated BrdU. The resulting DNA strand breaks are labeled the same way as for detection of apoptotic cells, with directly or indirectly labeled nucleotides, using exogenoeus terminal transferase. Labeling of apoptotic cells may be precluded by preincubation with dideoxynucleotide prior to photolysis, or they can be labeled with another color fluorochrome. Cell morphology is well preserved and the method is compatible with ethanol or formaldehyde fixation. It can be used to differentially identify the cell cycle position or DNA ploidy (by DNA content analysis) combined with the detection of apoptotic cells and BrdU incorporating cells, or alternatively, for identification of BrdU incorporating cells combined with mmunocytochemistry (Li et al., Exp. Cell Res., 22: 28-37, 1996; Li et al, Int. J. Oncol, 4:1157-61, 1994). Zbigniew Darzynkiewicz
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