Improved methods for immunohistochemical detection of BrdU in hard tissue

Journal of Immunological Methods
Akemi ShimadaAkira Nifuji

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 su...Continue Reading

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Citations

Mar 1, 2012·Histochemistry and Cell Biology·Akemi ShimadaAkira Nifuji
May 30, 2012·Microscopy and Microanalysis : the Official Journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada·Chuang-yu LinLi-tzu Li
Feb 7, 2014·Journal of Experimental Zoology. Part B, Molecular and Developmental Evolution·Anastasia Beiriger, Karen E Sears
Jun 4, 2014·Journal of Experimental Zoology. Part B, Molecular and Developmental Evolution·Sam VandenplasAnn Huysseune
Apr 20, 2017·Proceedings of the National Academy of Sciences of the United States of America·Subhash KulkarniPankaj J Pasricha
Dec 19, 2017·Journal of Orthopaedic Research : Official Publication of the Orthopaedic Research Society·Yugo MoritaShuichi Matsuda
Sep 16, 2010·Wound Repair and Regeneration : Official Publication of the Wound Healing Society [and] the European Tissue Repair Society·Gaoxing LuoJun Wu
Sep 1, 2021·Journal of the Korean Association of Oral and Maxillofacial Surgeons·Dong-Seok SohnYong-Suk Moon

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