Histochemistry
Cells were seeded at a density of 3 × 103 cells/ well in 48-well cell culture plates and permitted to grow to maturation for 3 (for RAW264.7 and MBM cells) or 7 days (for MC3T3 and ATDC5 cells) as described above. Thereafter, the herbal extracts (1, 10 and 100 μg/ml) or AD were added to the medium (0.5 ml/ well). After 3 days, the cells were stained for tartrate-resistant acid phosphatase(TRAP) [20] and alkaline phosphatase (ALPase) activity [21] and also stained with crystal violet [22], toluidine blue
[22] and alizarine red [23], as described previously, with slight modification.