Acetate → 4-Nitrophenol

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The scales treated by fixation were moved and weighed. After measurement, the scales were put into a 96-well microplate. To each well, 2001 of 100 mM acetate buffer (pH 5.3) containing 20 mM tartrate and 10 mM para-nitrophenol phosphate (substrate) was added. Reaction was performed at 25° C. for 20 minutes and subsequently terminated by the addition of a 3 M sodium hydroxide aqueous solution (50±1). Then, 1501 of the solution after the completion of reaction was transferred to another microplate. The amount of para-nitrophenol (p-NP) produced by TRAP was measured with a spectrophotometer (405 nm). The osteoclast activity was indicated as the amount of p-NP produced by the decomposition of para-nitrophenol phosphate per g of scale per minute.

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