Acid phosphatase assay kit

To evaluate the presence of Tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells and TRAP activity, the TRAP activity was confirmed using Sigma-Aldrich (Saint Louis, MO, USA) Acid Phosphatase Assay kit. RAW 264.7 cells were cultured in a 24-well plate at 5 × 10³ cells/well for 24 h and then treated with RANKL, LGS, and ODE for 7 days. Then, according to the manufacturer’s instructions, pick and dispense the supernatant from 96 wells, then dispense the substrate and let it react for 20 min in the incubator. Then add the stop solution, stop the reaction, and measure the absorbance at 405 nm. Quantification of TRAP activity was calculated according to the protocol of the Acid Phosphatase Assay kit (Sigma-Aldrich, Saint Louis, MO, USA). For TRAP staining, cell staining was performed using an acidic phosphatase solution containing sodium tartrate according to the manufacturer’s protocol after fixation with 4% formaldehyde for 15 min and after cell fixation. Stained cells were examined under a microscope and digital pictures were taken.

Tissues were harvested and lysed in immunoprecipitation assay buffer. Cellular debris was pelleted by centrifugation at 13 000 g for 30 minutes at 4°C. The total lysate protein were used to detect lysosomal enzyme activities using the Acid Phosphatase Assay Kit (Catalog Number CS0740; Sigma‐Aldrich), the β‐N‐Acetylglucosaminidase Assay Kit (Catalog Number CS0780; Sigma‐Aldrich) and Cathepsin D Activity Assay Kit (ab65302; Abcam).

Mouse femoral bones were fixed in 4% PFA for 2 h at 4 °C, decalcified with 0.5 M EDTA (Sigma-Aldrich) for 4 weeks, and decalcified bones were immersed in a 20% sucrose and 2% polyvinylpyrrolidone (Sigma-Aldrich) solution for 1 week.
For immunostaining, the bones were embedded and frozen in OCT, and 6-μm-thick sections were cut along the longitudinal axis of the femur. Bone sections were blocked in 5% bovine serum albumin and 0.3% Triton X-100 for 1 h, and incubated with antibodies (1:300 for primary antibodies, 1:1000 for secondary antibodies). Nuclei were counterstained with DAPI. The antibodies were as follows: anti-CD3 (abcam, Cambridge, MA), anti-RANKL (abcam), Alexa Fluor 555 Donkey Anti-Rabbit IgG H&L, and Alexa Fluor 488 Goat Anti-Rat IgG H&L (abcam).
To detect osteoblasts and osteoclasts, the tissues were dehydrated in 75% ethanol overnight, then sequentially in 85% ethanol, 95% ethanol, and 100% ethanol for 1 h each. The femoral bones were treated with xylene twice for 15 min, then embedded in paraffin, and 5-μm-thick sections were taken. Morphological analysis of osteoblasts was performed by standard hematoxylin and eosin (H&E) staining. TRAP was stained using an acid phosphatase assay kit (Sigma-Aldrich), and TRAP-positive osteoclasts were examined at 20× magnification.