Abstract：
:Bromodeoxyuridine (BrdU) is used to label synthesizing DNA and to chase label-retaining cell (LRC). As stem cells divide slowly in adult tissues, they can be visualized as LRCs. In order to identify LRCs in hard tissue, we examined optimal conditions of fixation, demineralization, and DNA denaturation/antigen retrieval for immunohistochemistry of BrdU in hard tissues including bone, tooth, and periodontal ligament. Mice were subcutaneously injected with BrdU (50 microg/g body weight) twice a day from the postnatal day 11 to day 15 and sacrificed at 2 h after the last injection. Dissected maxillae were fixed (Bouin's solution or 4% paraformaldehyde), demineralized (Morse's solution or EDTA), and embedded in paraffin. Antigen retrieval procedures were performed before incubation with primary antibody. When sections were treated with HCl for DNA denaturation, the staining intensity of BrdU positive cells was not affected by difference of fixatives. Higher sensitivity was obtained by demineralization with Morse than with EDTA. Although heat-induced antigen retrieval techniques in citrate buffer (pH 6.0) showed as well or better sensitivity than acid pretreatment, heating caused tissue damage specifically to tooth dentine and the surrounding tissue. When the LRCs at four weeks after the last injection of BrdU were compared, much more LRCs were observed in specimen demineralized with Morse than with 10% EDTA. Our data suggest that demineralization with Morse with Bouin fixative plus HCl pretreatment gives rise to the optimal results for BrdU immunodetection in hard tissue.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Shimada A,Shibata T,Komatsu K,Nifuji Adoi
10.1016/j.jim.2008.07.013subject
Has Abstractpub_date
2008-11-30 00:00:00pages
11-6issue
1eissn
0022-1759issn
1872-7905pii
S0022-1759(08)00234-2journal_volume
339pub_type
杂志文章abstract：:Three different methods for the radioiodination of surface proteins in trypanosomatids have been comparatively analyzed. Data interpretation in the lactoperoxidase-mediated method was impaired by autoiodination of the enzyme and its tight retention by the cells. In the enzymobeads method, iodinated enzymes were releas...
journal_title：Journal of immunological methods
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journal_title：Journal of immunological methods
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journal_title：Journal of immunological methods
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journal_title：Journal of immunological methods
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journal_title：Journal of immunological methods
pub_type： 杂志文章
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journal_title：Journal of immunological methods
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journal_title：Journal of immunological methods
pub_type： 杂志文章
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journal_title：Journal of immunological methods
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journal_title：Journal of immunological methods
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journal_title：Journal of immunological methods
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journal_title：Journal of immunological methods
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journal_title：Journal of immunological methods
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journal_title：Journal of immunological methods
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journal_title：Journal of immunological methods
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journal_title：Journal of immunological methods
pub_type： 杂志文章
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journal_title：Journal of immunological methods
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