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Briefly, adherent and non-adherent osteoclasts were collected, fixed with 4% paraformaldehyde and cytospun into glass slides. Apoptosis was identified on the basis of characteristic changes nuclear morphology using TUNEL staining as previously described [40]. Cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI, 1 µg/ml) for 3 min prior to analysis by fluorescence microscopy. Cells were scored as apoptotic on the basis of nuclear morphology. An average of five microscopic fields per treatment group was analysed (×200 magnification).
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