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
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
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Summary |
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Key Words: thymidine analog double labeling cell cycle S-phase
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Introduction |
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Materials and Methods |
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DNA Double Labeling
Male mice aged 4 or 10 weeks (Slc/ICR) were injected intraperitoneally with 50 mg/kg body weight of BrdU (TCI Tokyo Kasei Kogyo Co., Ltd.; Tokyo, Japan) dissolved in physiological saline. After 1 hr, they received an injection of 50 mg/kg of CldU (ICN Biomedicals; Aurora, OH). After an additional hr, the animals were sacrificed under deep anesthesia, and the small intestines were removed, fixed in 70% ethanol, and routinely processed to 5-µm-thick paraffin sections.
To establish the specificity of the antibody, mice injected with either CldU or BrdU were prepared and their intestines were used for examination of binding.
Immunohistochemistry
After deparaffinization and rehydration through graded series of ethanol, sections were pretreated in a microwave oven in sodium citrate buffer (pH 6.0) for 5 min at 100C. After they were cooled down in NaCl-TB (300 mM NaCl in 50 mM Tris buffer, pH 7.4) for 5 min, they were incubated with 1% skimmed milk in NaCl-TB for 5 min to block nonspecific binding.
For double staining with DAB and fluorescent dye, sections were incubated with CldU antibody (10 µg/ml) overnight. After they were exposed to biotinylated goat anti-rabbit IgG (Lab Vision Corporation; Fremont, CA) for 60 min, they were incubated with 1:2000 diluted horseradish peroxidase avidin D (Vector Laboratories; Burlingame, CA) for 60 min, then 0.02% of 3,3'-diaminobenzidine tetrahydrochloride (DAB, Dojinkagaku, Kumamoto, Japan) in 100 mM imidazole-HCl solution, pH 7.4, with 300 mM NaCl and 0.006% of H2O2 for 15 min. After they were washed, they were treated again in a microwave oven in sodium citrate buffer for 5 min at 100C and incubated with Br-3 antibody (1:10000) overnight, followed by Alexa Fluor 594 goat anti-mouse IgG (1:400; Molecular Probes, Eugene, OR) in NaCl-TB with skimmed milk for 3 hr. Finally they were mounted in mounting medium containing DAPI (Vector Laboratories).
For double immunofluorescent staining, after pretreatment in a microwave oven, sections were incubated with CldU (10µg/ml) and Br-3 (1:10000) antibodies overnight. Then the sections were incubated with a mixture of Alexa Fluor 594 goat anti-rabbit IgG (1:400) and Alexa Fluor 488 goat anti-mouse IgG (1:400) for 3 hr and mounted in the medium containing DAPI. Sections from mice injected with either CldU or BrdU were also treated with the same method to determine reactivities of antibodies used in the present study.
All stained sections were examined under a fluorescent microscope equipped with a digital camera (Nikon DXM 1200; Tokyo, Japan). Photographs of DAB stained sections were treated with a computer program (Photoshop) to reverse the tone of the pictures and merged with corresponding fluorescence images.
DNA Double Labeling of Cultured Cells
Neural progenitor cells were cultured according to the method described by Shinohara et al. (2004). Dissociated cells (45 x 105/dish) were seeded onto 35-mm dishes with 12 x 9 mm coverslips precoated with poly-L-lysine (Sigma) and propagated in serum-free medium (SFM) with fibroblast growth factor-2 (FGF-2) for 24 hr. After 24 hr, some cultured cells were incubated in SFM with FGF-2 and BrdU (10 µM) for 60 min. After the incubation, the culture medium was exchanged to SFM with FGF-2 and CldU (10 µM), and the cells were cultured for another 60 min (pulse-chase-pulse-chase experiment). Other cultured cells were incubated in SFM with FGF-2 and BrdU (10 µM) for 60 min. Then CldU (10 µM) was added to the culture medium and the cells were incubated for another 60 min (no-chase experiment). Finally, the cultured cells were fixed in 70% ethanol for 20 min and then stored in PBS with sodium azide until use.
The methods of double immunostaining were the same as those of histological sections.
Estimation of Cell Cycle Length
To calculate the duration of S-phase (Ts) and the length of a cell cycle (Tc), we counted the number of nuclei of the intestinal epithelial cells that were stained only with Br-3 antibody, and with both Br-3 and CldU antibodies, respectively, and the total number of nuclei comprising the proliferating zone in the same section. The duration of S-phase (Ts) was calculated as the number of double stained cells/Br-3 stained cells x t, where t is the time interval between injection of the two labels (in this study 1 hr). The length of a cell cycle (Tc) was expressed as Ts/CldU labeling index (LI) x GF, where GF is the "growth fraction," assumed to be 100% in this case. The CldU LI is the percentage of CldU-labeled nuclei among the DAPI positive nuclei.
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Results |
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Discussion |
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Identification of cells in early S-phase has been performed in cultured cells (Nakayasu and Berezney 1989; O'Keefe et al. 1992
; Manders et al. 1992
, 1996
; Ma et al. 1998
). In the present study, identification of those cells in tissue sections was attempted. It is known that eukaryotic chromosomal DNA is divided into hundreds to thousands of independent subunits of replication named replicons (Hand 1978
). When BrdU and CldU were incorporated into a replication site simultaneously, DAB pigmentation due to CldU staining can quench the colocalized fluorescence due to BrdU, while when they were incorporated sequentially, fluorescence of BrdU will not be quenched by subsequent DAB staining because the replication sites moved during the 1 hr interval between the administrations. In the present study, cells entered the S-phase during the second 60 min were expected to incorporate both BrdU and CldU simultaneously and will be stained only with DAB (Figure 4). In practice, cell nuclei stained only with DAB were found and the staining pattern was diffuse suggesting many replication sites. In previous reports on cultured cells, replication sites in early S-phase consisted of many small domains scattered throughout the nucleoplasm, whereas in late S-phase they were larger in size and smaller in number (Manders et al. 1992
,1996
; Ma et al. 1998
). An electron microscopic study revealed that cells in early S-phase display a diffuse pattern of labeling with many spots, whereas cells in late S-phase show labeling confined to large domains, often at the periphery of the nucleus or associated with nucleolus (Jaunin et al. 1998
). Thus, the present observation was in good agreement with these reports on cultured cells (Figure 4 and Figure 5).
Considering the good correlation between the present double-labeling study and the previous ones, we can conclude that our double labeling technique using CldU and BrdU in combination with DAB and fluorescent staining enabled to identify cells in early S-phase in tissue sections. By shortening the interval between the 2nd injection and the fixation, it will be possible to identify the cells in very early S-phase.
In embryos, cell nuclei of the neuroepithelium, a kind of pseudostratified epithelium, are positioned in a variety of levels and were reported to change the level depending on their cell kinetics (Fujita 1962). In the present study, most of nuclei of the intestinal columnar epithelium are positioned in basal level, while nuclei stained only with Br-3 antibody were frequently found in apical level (Figure 4). Because the cells stained only with Br-3 antibody were thought to have finished DNA synthesis during the first 60 min, the displaced nuclei stained only with Br-3 antibody suggest intracellular movement of nuclei in the G2- or M-phase in columnar epithelium. Because the displaced nuclei were frequently not oval in shape, they may be in M-phase.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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