Detection of cell cycle stages in situ in growing cell populations

Microscopic in situ detection of the cell cycle stages is based on immunocytochemical techniques and allows one to determine the stage of the cell cycle of individual cells. Based on pKi-67 immunostaining, one can distinguish between proliferating and quiescent cells and identify cells in early G1. Denaturation is a critical step of detection because it can negatively affect preservation of cell morphology. In particular, incubation of cells in 2N HCl for 30-60 min strongly impairs cell and nuclear morphology and, therefore, should be avoided in assays where the conservation of cell morphology is important. Epitops of Ki-67 protein is stable and the protein can be detected after 0.1N HCl and denaturation steps with heat denaturation or DNase. Therefore, pKi-67 immunostaining can be performed either simultaneously or after detection of incorporated halogenated thymidine analogs. The antibody to BrdU supplied with the kit contains nucleases generating single-stranded DNA fragments in the nucleus. © 2006 Elsevier Inc. All rights reserved.