Trap alkaline phosphatase alp stain kit

Osteoclastogenesis in bone marrow macrophage cultures was determined as we described [20] (link). In brief, four-week-old male C57BL/6 mice were sacrificed by cervical dislocation and bone marrow cells in the femurs and tibiae were harvested by flushing out with phosphate-buffered saline using a 27 gauge needle. The cells were then incubated in α-MEM with 30 ng/ml macrophage-colony-stimulating factor (M-CSF) (R&D System, Minneapolis, MN) for 24 h. Non-adherent cells were transferred to 48-well plates (1 × 10⁵ cells/well) and treated with suboptimal dose of 30 ng/ml M-CSF and 10 ng/ml receptor activator of NF-κB ligand (RANKL) (PEPROTECH, Rocky Hill, NJ) in the absence or presence of 4T1 CM (20%, v/v) and with or without the TLR4 antagonist TAK-242 or RAGE antagonist FPS-ZM1. Following five days of culture, the cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP) using a TRAP/alkaline phosphatase (ALP) Stain Kit (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The number of TRAP-positive multinucleated osteoclast-like cells (TRAP⁺ MNOCs) (nuclear number > 3) in each well were counted under a microscope.

Twelve weeks after surgery, the rats were euthanized by an overdose of isoflurane and then transcardially perfused with cold saline, followed by 4% paraformaldehyde in phosphate-buffered saline, pH 7.4. The implants were resected along with the surrounding bone and tissue and fixed with 4% paraformaldehyde in phosphate-buffered saline. The samples were then decalcified with 10% ethylenediaminetetraacetic acid, embedded in paraffin, and cut in the coronal plane at 3-μm thickness. For morphological evaluation, the sections were stained with hematoxylin and eosin. To confirm the presence of osteoclasts, the sections were stained using a tartrate-resistant acid phosphate (TRAP)/alkaline phosphatase (ALP) stain kit (294-67001, Wako, Japan) without double staining by ALP. To evaluate angiogenesis, the sections were incubated with a rabbit anti-CD34 antibody (1:200, ab81289, Abcam) at 4 °C overnight. Immunohistochemistry was performed with the avidin–biotin technique, and nuclei were counterstained with hematoxylin.