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Discrimination of Cell Nuclei in Early S-phase, Mid-to-late S-phase, and G₂/M-phase by Sequential Administration of 5-Bromo-2'-Deoxyuridine and 5-Chloro-2'-Deoxyuridine

Kumiko Yamada, Reiji Semba, XiaoHui Ding, Ning Ma and Masato Nagahama

Department of Anatomy, Mie University School of Medicine, Mie, Japan (KY,XHD,NM,MN); College of Nagoya Women's University, Aichi, Japan (KY); and Institute for Developmental Research, Aichi Human Service Center, Aichi, Japan (RS)

Correspondence to: Ms. Kumiko Yamada, College of Nagoya Women's University, 3-40 Shioji cho, Mizuho-ku, Nagoya, Aichi, 467-8610, Japan. E-mail: kyamada{at}nagoya-wu.ac.jp

5-Bromo-2'-deoxyuridine (BrdU) and 5-chloro-2'-deoxyuridine (CldU) were sequentially administered intraperitoneally into mice at 1-hr intervals. After one additional hr, the small intestines were resected, fixed, and embedded in paraffin. In histological sections stained with monoclonal antibody Br-3 reactive to both BrdU and CldU, and CldU antibody reactive only to CldU, three types of staining could be identified in the proliferating zone. Cells with nuclei stained only with Br-3 antibody were estimated to have completed DNA replication during the first 1 hr and were fixed in G₂/M-phase. Those nuclei were frequently found in apical areas of the simple columnar epithelium of the intestine, whereas other nuclei were located basally in the cells. This observation suggested intracellular movement of cell nuclei in G₂/M-phase. Identification of cells in early S-phase became possible using these antibodies in combination with DAB and fluorescence stainings. Replication sites in early S-phase nuclei were found to be numerous, whereas in late S-phase they were larger in size and much smaller in number. (J Histochem Cytochem 53:1365–1370, 2005)

Key Words: thymidine analog • double labeling • cell cycle • S-phase

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