Bones (femurs and L2 vertebrae) were fixed for 24 h in 10% (vol/vol) Millonig’s formalin, decalcified in 14% (wt/vol) EDTA for 1 week, embedded in paraffin, and then 5 µm longitudinal sections were cut. After removal of paraffin and rehydration, the sections were stained for TRAP activity and counterstained with methyl green. Quantitative histomorphometry to determine osteoclast number was performed on the TRAP-stained sections using a computer and digitizer tablet (OsteoMetrics, Decatur, GA) interfaced to a Zeiss Axioscope (Carl Zeiss, Thornwood, NY) attached with a drawing tube. In L2 vertebrae, all cancellous bone was analyzed with the exception of the region within two visual fields of the growth plates and any trabeculae that were in direct contact with cortical bone. In femurs, cancellous bone analysis was performed beginning two visual fields below the growth plate and extended the entire remaining area of cancellous bone in the samples from RANKLf/f mice. Since the cancellous bone of the conditional knockout mice was much more extensive, a region similar in area to the RANKLf/f controls was measured and averaged approximately 3 mm2. Histological measurements of the endocortical bone of the femur also began two visual fields below the growth plate and were continued to the diaphysis, with the diaphysis defined as the midway point between the ends of the femur.
Computer and digitizer tablet
Manufactured by OsteoMetrics
Sourced in United States
The computer and digitizer tablet is a core component of the OsteoMetrics lab equipment. It serves as the central processing and input device for the system. The computer provides the computational power and interface for data analysis and management, while the digitizer tablet allows for the direct input and recording of measurements and data.
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Lab products found in correlation
5 protocols using computer and digitizer tablet
1
Quantitative Histomorphometric Analysis of Osteoclasts
2
Histomorphometric Analysis of Lumbar Vertebrae
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The L5 lumbar vertebrae were fixed with 10% formalin in phosphate-buffered saline (pH 7.4), and embedded undecalcified in methyl methacrylate. The histomorphometric examination was performed using a computer and digitizer tablet (OsteoMetrics Inc., Atlanta, GA) interfaced to a Zeiss Axioscope (Carl Zeiss, Inc., Thornwood, NY) with a drawing tube attachment. The cancellous measurements were two-dimensional, confined to the secondary spongiosa, and made at a magnification of 400. The region of interest was selected by a boundary beginning 0.5 mm proximal to the midpoint of the growth plate, non-inclusive of cortical bone, and extending proximally for a total area of approximately 2.8 mm2. The terminology and units used are those recommended by the Histomorphometry Nomenclature Committee of the American Society for Bone and Mineral Research [18 (link)].
3
Histological Analysis of Osteoclast Formation
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We fixed the lumbar vertebrae (L2–L4) for 24 h in 10% Millonig’s formalin and decalcified in 14% EDTA for 1 week. Vertebrae were then embedded in paraffin, and 5-μm longitudinal sections were obtained. After de-paraffinization and rehydration, sections were incubated with 0.2 M acetate buffer containing 10% (vol/vol) naphthol-AS-BI-phosphate at 37 °C for 30 min. The sections were then incubated with pararosaniline chloride (1.5 g ml−1) for 5 min and counterstained with 0.1% fast green. In some experiments (indicated in the text), we embedded undecalcified vertebrae in methyl methacrylate and 4- to 8-μm longitudinal sections were obtained. Before staining, we eluted the plastic with 2-methoxyethyl acetate. Decalcified and undecalcified TRAP-stained sections, counterstained with toluidine blue, were used for osteoclast enumeration. Osteoclast counts were performed using a computer and digitizer tablet (OsteoMetrics) interfaced to a Zeiss Axioscope (Carl Zeiss) with a drawing tube attachment. The numbers of TRAP-positive multinucleated cells on the cancellous perimeter (osteoclast number) were measured directly. Terminology recommended by the Histomorphometry Nomenclature Committee of the American Society for Bone and Mineral Research was used in this study68 (link).
4
Quantitative Bone Histomorphometry Analysis
5
Histomorphometry and Apoptosis Analysis of Distal Femur
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