Images were obtained by Nikon DS-Fi2 color camera (Nikon Instruments, Melville, NY, USA) using UV and Cy3 filters and added in Image J [ Ot, and total Ot were quantified in cancellous bone within 1 mm into the metaphysis from the proximal EMB and in cortical bone within 4–10 mm from the proximal EMB.The means were calculated for each animal with respect to the parameters above and used for comparison between the groups.Nonspecific rabbit Ig G served as a negative control for m TRAP, while TRAP enzyme was inactivated using 100 μM molybdate before adding ELF97 to evaluate the background fluorescence.
Intracellular TRAP was located to electron-dense vesicles with similar morphology in both cell types.
Immunogold labeling was performed as earlier described . Ar with an interclass correlation of Semiquantitative measurements were performed on sections subjected to in situ hybridization or immunofluorescence in order to estimate the distance from bone surfaces or bone remodeling surfaces toward osteocytes expressing TRAP m RNA, TRAP protein (m TRAP), or TRAP enzyme activity in cancellous and cortical bone.
Nonspecific rabbit Ig G served as a negative control. All animals subjected to TRAP m RNA in situ hybridization as well as three animals from each group in both cancellous and cortical bone subjected to fluorescence-based staining with m TRAP and the fluorescence substrate ELF97 were analyzed.
Ovx-D increased osteoclast activity (]—has been an established marker for osteoclasts and bone resorption for more than 50 years.
TRAP is synthesized as a relatively inactive proenzyme (monomeric TRAP [m TRAP], loop-TRAP, serum TRAP 5a), and proteolytic cleavage by members of the cathepsin family or other proteinases increases the catalytic activity at least tenfold  have shown that the serum activity of TRAP 5b is significantly elevated in patients with osteoporosis and negatively correlated with bone mineral density (BMD).
In the experimental rickets group, commercially available kits were used for determination of osteoclast-derived C-telopeptide fragments of collagen type I (CTX) (Rat Laps™ EIA; Immunodiagnostic Systems, Tyne and Wear, UK) and osteoclast-derived TRAP 5b (Rat TRAP™ Assay, Immunodiagnostic Systems).