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DOI: 10.1369/jhc.4A6285.2004
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Journal of Histochemistry and Cytochemistry
Volume 52 (11): 1503-1509, 2004
Copyright ©The Histochemical Society, Inc.

Characterization of a Monoclonal Antibody, HTA28, Recognizing a Histone H3 Phosphorylation Site as a Useful Marker of M-phase Cells

Akihiro Hirata, Ken-ichi Inada, Tetsuya Tsukamoto, Hiroki Sakai, Tsutomu Mizoshita, Tokuma Yanai, Toshiaki Masegi, Hidemasa Goto, Masaki Inagaki and Masae Tatematsu

Division of Oncological Pathology, Aichi Cancer Center Research Institute, Nagoya, Japan (AH,KI,TT,HS,TM,MT); Department of Veterinary Pathology, Gifu University, Gifu, Japan (AH,HS,TY,TM); and Division of Biochemistry, Aichi Cancer Center Research Institute, Nagoya, Japan (HG,MI)

Correspondence to: Masae Tatematsu, Div. of Oncological Pathology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan. E-mail: mtatemat{at}aichi-cc.jp

Mitosis is a valuable indicator of active tissue proliferation but, other than morphological characteristics, there have hitherto been no markers available to detect only M-phase cells. However, a newly established monoclonal antibody (MAb), HTA28, recognizing histone H3 (H3) harboring phosphoserine 28, allows visualization with mitotic chromosomal condensation. In this study we investigated the use of HTA28 for immunohistochemical (IHC) detection of M-phase cells in the regenerating rat liver after partial hepatectomy (PH). Groups of three to five rats were sacrificed at intervals up to 72 hr after PH and proliferation was then assessed by IHC staining using HTA28 and other markers. The temporal pattern of the HTA28 staining index (SI) was very similar to that for the mitotic index (MI), also showing similarities to the bromodeoxyuridine (BrdU) labeling index (LI) with a time lag. The HTA28 SI proved to be higher than MI at every time point in line with HTA28 immunoreactivity maintained for all stages of M-phase. The spatial distribution of HTA28-positive cells corresponded with those of other proliferative cell markers. These therefore provide strong evidence for the applicability of HTA28 as an M-phase marker. We also showed that antigenicity for HTA28 is lost if tissue is not immediately fixed after sampling. (J Histochem Cytochem 52:1503–1509, 2004)

Key Words: M-phase cell • cell proliferation • immunohistochemistry • histone phosphorylation • rat • liver • partial hepatectomy


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