Improved methods for immunohistochemical detection of BrdU in hard tissue

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 µg/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.