Triple S-Phase Labeling of Dividing Stem Cells

Marking replicating DNA with multiple labels presents the possibility of revealing new features and mechanisms of DNA synthesis and cell division; however, progression beyond double labeling has been hampered by cross-reactivity of label detection and scarcity of appropriate labels. Here, we present a method for triple S-phase labeling of the dividing cells, with a fourth label used to mark cells actively engaged in cell-cycle progression (e.g., using Ki67) or to phenotype the dividing cells or their progeny (e.g., using a GFP-expressing lineage reporter transgene). We apply this method to determine the parameters of neural stem cell division in the adult brain, to birth date up to four cohorts of dividing cells, and to reveal patterns of stem cell division in non-neural tissues.