Vertebral and tibial bones of older pups or adult mice were decalcified for 7 days in 14% (wt/vol) EDTA (pH adjusted to 7.2 by addition of ammonium hydroxide) and then embedded in paraffin. Vertebral bones (tails) were cut into 5-μm sections, tibiae into 8-μm sections, respectively. For morphological assessment, tissue sections were stained with hematoxylin and eosin. Alcian blue staining was used to visualize the cartilaginous components of the epiphyseal growth plate. In order to evaluate trabecular bone parameters and the amount of osteoclasts, tissue sections were stained for tartrate-resistant acid phosphatase using the leukocyte acid phosphatase (TRAP) kit 387A (Sigma-Aldrich, St. Louis, MO, United States) according to the manufacturer’s instructions. All parameters were quantified by digital image analysis (OsteoMeasure; OsteoMetrics, Decatur, GA, United States).
Trap kit 387a
Manufactured by Merck Group
Sourced in United States
The TRAP) kit 387A is a laboratory equipment product manufactured by Merck Group. It is designed to perform a specific function, but a detailed and unbiased description cannot be provided while maintaining conciseness and avoiding interpretation or extrapolation.
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6 protocols using trap kit 387a
1
Histomorphometric Analysis of Bone in Mice
2
Osteoclastogenesis Analysis of Murine Bone Marrow
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Primary bone marrow osteoclastogenesis analysis was performed with bone marrow from 4-to-6-week-old BALB/c mice. Bone marrow cells were harvested and plated in basal culture medium overnight. The next day, non-adherent cells were added at 5 × 105 per well to 24-well plates that were previously seeded with either control or indicated tumour cells supplemented with 25 ng ml−1 RANKL(PeproTech) and 25 ng ml−1 M-CSF(PeproTech). Medium was changed every 3 days. Cancer cells were treated with 10 ng ml−1 TGFβ for 72 h, and the resultant conditioned medium were mixed with α-MEM at 1:9 ratio for bone marrow culturing. TRAP staining was performed with a TRAP kit (387A, Sigma-Aldrich) on day 10–12. Osteoclasts were defined as TRAP-positive cells containing more than three nuclei.
3
Osteoclast Differentiation and Visualization
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After the completion of osteoclast differentiation, osteoclasts were observed under a light microscope, and cells were fixed for 30 min using 4% paraformaldehyde dissolved in PBS at room temperature and further using a Leukocyte acid phosphatase (TRAP) kit (387-A; Sigma–Aldrich) differentiated osteoclasts were stained according to the instructions. Images of multinucleated TRAP-positive cells were captured with an EVOS FL auto microscope33 (link).
4
Osteoclast Differentiation and Inhibition
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BMMs were cultured in the presence of RANKL and M-CSF, following BMMs were treated with A438079 (ab120413; Abcam) for 12 h, after 5 days, the cells were fixed in 10% paraformaldehyde for 10 min. After washing with PBS, the cells were stained using a TRAP-Kit 387A (Sigma-Aldrich; MerckKGaA) according to the manufacturer's instructions. TRAP-positive mature osteoclasts having more than three nuclei were observed and counted using a light microscope (LEICA DMI 3000B; Leica Microsystems GmbH).
5
Osteoclast Quantification in Murine Arthritis
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Sections of knee joints obtained from C57BL/6 mice at 7 days after i.a. mBSA administration were stained according to tartrate-resistant acid phosphate (TRAP) kit 387A (Sigma-Aldrich, St. Louis, MO, USA). TRAP was used as a histochemical marker of osteoclasts in 5 µm sections (Ballanti et al. 1997) and TRAP-positive cells appeared as dark purple. Osteoclasts were identi ed as TRAP-positive multinucleated cells and counted in the joint growth area (femur). The result was expressed as the number of TRAP-positive cells.
6
Osteoclast Generation from Murine BMCs
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Bone marrow cells (BMCs) from 8-week-old male C57BL/6 mice were obtained by ushing tibias and femurs with α-MEM (Thermo Fisher Scienti c, Waltham, MA, USA) supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL). Cells were transferred to 10 cm Petri dishes and then cultured in the presence of M-CSF (30 ng/mL; R&D Systems, Minneapolis, MN, USA) for 3 days. After that, oating cells were discarded, and the adhering cells were classi ed as BMMs (bone marrow macrophages). To generate osteoclasts, BMMs were cultured in 96-well plates (2 × 10 4 cells/well) at 37°C/5% CO 2 in the presence of osteoclastic media, which consists of M-CSF (30 ng/mL) and RANKL (10 ng/mL), with different concentrations of methyl gallate (0, 3, 10, 30, 100 µM). After 3 days, the cells were stained according to TRAP kit 387A (Sigma-Aldrich, St. Louis, MO, USA). TRAP-positive multinucleated cells with more than three nuclei were identi ed as osteoclasts. Mature osteoclasts were counted and represented as multinucleated TRAP + cells/well. To determine the osteoclast area, we analyzed the images using ImageJ software.
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