Femora were fixed in 4% paraformaldehyde, decalcified in EDTA, embedded in paraffin and 3μm microtome sections (parasagittal plane) cut, dewaxed, reacted for TRAP and counterstained with Mayer's hematoxylin (Sigma-Aldrich). Osteoclast number (Oc.N./BS), and osteoclast surface (Oc.S/BS) were examined in two regions of the bone, the growth plate/primary spongiosa and in mature trabecular bone. For the latter, a region of interest 200 × 200 μm2 located 0.4mm below the distal growth plate was employed. These analyses were performed using Osteomeasure histomorphometry software (Osteometrics, Atlanta, GA) [25 (link)].
Osteomeasure histomorphometry software
Manufactured by OsteoMetrics
Sourced in United States
OsteoMeasure is a histomorphometry software designed for the analysis and quantification of bone tissue samples. The software provides a set of tools for the measurement and assessment of various bone parameters, including bone volume, trabecular thickness, and connectivity.
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Lab products found in correlation
3 protocols using osteomeasure histomorphometry software
1
Osteoclast Quantification in Bone Sections
2
Histomorphometric Analysis of Tumor Bone Invasion
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Skeletal tissues were fixed in 10% buffered formalin, decalcified in 14% EDTA, embedded in paraffin, and sectioned at 4 μm along the midsagittal plane. Sections were stained with hematoxylin and eosin (H&E) and analyzed for tumor infiltration of bones stereologically using an Olympus BX40 microscope (Olympus, Center Valley, PA, USA) connected to desktop computer running the OsteoMeasure histomorphometry software (Osteometrics, Atlanta, GA, USA) [24 (link), 26 (link), 61 (link)]. Consecutive sections of tibiae and femora were stained for tartrate-resistant acid phosphatase (TRAP) and TRAP+ multinucleated cells (nuclei ≥ 3) counted as osteoclasts. Soft tissues were also embedded in paraffin and tumor volume assessed as described above. In all cases, at least three sections from non-consecutive levels were quantified and tumor volume calculated as tumor area/total bone marrow cavity area is expressed as mean ± SEM.
3
Histomorphometric Analysis of Mineralized Tissue
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Following micro-CT analysis, the samples were rinsed 3 times with PBS and demineralized in formic acid. The specimens were dehydrated in a graded ethanol series for paraffin embedding and preparation of sagittal sections (23) . The sample blocks were oriented mesio-distally and sectioned using an automatic rotary tissue microtome (Leica RM 2255, Leica Biosystems) set at 3 µm thickness. The sections were subjected to either hematoxylin and eosin (H&E) or Masson's trichrome staining. The mineralized volume/tissue volume was measured using OsteoMeasure histomorphometry software (Osteometrics, Atlanta, GA, USA). In brief, the mineralized area was selected, and its size relative to the total tissue volume was calculated (24) . Each slide was subjected to measurement in 5-8 different areas at ×10 magnification.
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