Tracp and alp double stain kit

Since alkaline phosphatase is an enzymatic reaction, fixation and long incubation time in chemical solution will affect the result. To address this problem, we perform Kawamoto’s film method using fresh and unfixed samples (Kawamoto, 2003 (link)). Briefly, fish at 3 months were anesthetized and then cut into three pieces. The cut samples were embedded into embedding medium before being soaked in cooled isopentane with liquid nitrogen. Using cryofilm tape, samples were sectioned at 20 μm thickness. Sections were fixed with 4% PFA for 5 min then washed three times using PBS. Takara TRACP and ALP double-stain Kit was used for the detection of alkaline phosphatase according to manufacture. Staining was performed at room temperature for 30 min.

Calvarial cells (POBs) isolated from neonatal Arl6ip5^Δ2/Δ2 or wild-type mice were co-cultured with non-adherent bone marrow cells. POBs at a density of 1 × 10³ cells per well in 48-well plates were co-cultured with 2 × 10⁴ per well non-adherent bone marrow cells isolated from wild-type mice for 7–9 days in α-MEM containing 10% FBS and 100 nM PTH. One-half of the medium was replaced with fresh medium and PTH every 3 days. After 9 days co-cultures, cells were fixed and stained for TRAP using TRACP and ALP double-stain Kit (TaKaRa Bio Inc.). The osteoclast with three or more nucleus was calculated with Image J software. For resorption analysis, 1 × 10⁴ Raw264.7 cells were seeded into the 24-well culture plate that contained 2 × 10³ POBs and dentine slices (Immunodiagnostic Systems PLC, Boldon, UK), 100 nM PTH was added the next day and culture for 5–6 days with regular medium changed. Slices were incubated with 0.25 M ammonium hydroxide and sonicated for several times. The slices were stained with 0.1% toluidine blue in 0.5% sodium tetraborate for 5 min, washed with water and air dried before photographs were taken by reflected light microscope. The resorpted areas were measured by Image J software.