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The cloned insert was sequenced to establish the orientation (Seqlab, Göttingen, Germany).

A digoxigenin (DIG)–conjugated complementary RNA probe was synthesized using T7 or Sp6 polymerase to yield the probe in the sense or antisense direction (DIG-labeling kit; Roche Diagnostics, Oslo, Norway).

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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).

Serum was analyzed in all animals, and the CTX/TRAP 5b ratio, as a parameter for osteoclast activity [] was amplified by conventional PCR using c DNA from rat bone and oligonucleotide forward and reverse primers: rn 5′-ACGCCAATGACAAGAGGTTC-3′, rn 5′-ACATAGCCCACACCGTTCTC-3′ (Life Technologies, Carlsbad, CA, USA) and cloned in a Dual Promoter TA Cloning Kit (Life Technologies).

Micrographs from 10–20 osteoblasts and osteocytes were randomly sampled from each animal. Longitudinal tibia mid-diaphyseal sections from Ovx-D and sham animals at the same bone level were subjected to conventional hematoxylin–eosin–saffron (HES) staining.

An osteoblast was defined as a mononuclear cell attached to osteoid with prominent endoplasmatic reticulum (ER) and Golgi complexes. Ar) was measured in each osteoblast and osteocyte and the mean of the ratios (TRAPv. The osteocyte lacunar area was measured within 1 mm at three discrete sites separated by 1 mm in a cross-sectional manner.

Tartrate-resistant acid phosphatase (TRAP) is known as an osteoclast marker, but osteoblasts and osteocytes in the vicinity of bone remodeling sites also express TRAP.

Cell culture studies suggest that osteoblasts endocytose osteoclastic TRAP for inactivation.

To evaluate whether changes in osteoclast activity could alter TRAP expression in osteoblasts and/or osteocytes in vivo, we studied the ovariectomized and vitamin D-deficient rat (Ovx-D) and rats healing from rickets.

Bone sections were analyzed for TRAP gene expression by in situ hybridization, TRAP protein by immunogold labeling, and TRAP enzyme activity using the fluorescent substrate ELF97.

Longitudinal sections from the tibial diaphysis (Ovx-D/sham) and femoral diaphysis (experimental rickets) were subjected to hybridization following our established protocol [ osteocytes were quantified in cortical bone within 4–10 mm from the proximal EMB by point counting in a squared grid.

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