Fast red violet lb stain

The 6-well plates were removed from the incubator and the cells carefully washed with 500 µL PBS. The cells were fixed with 400 µL 10% glutaraldehyde solution. The glutaraldehyde solution was removed, and the cells were washed with 400 µL PBS at least three times. The cells were coated with 400 µL freshly prepared TRAP solution (0.3 mg Fast Red Violet LB stain (F-3381, Sigma-Aldrich, St. Louis, MO, USA) per mL TRAP Buffer (50 mL 0.1 M Acetate Buffer, 10 mL 0.3 M Sodium Tartrate, 1 mL 10 mg/mL Naphtol AS-MX Phosphate (N-5000, Sigma-Aldrich, St. Louis, MO, USA), 100 µL Triton X-100, 38.9 mL H₂O_dd) and incubated at 37 °C for 15 min. Again, the cells were washed twice with 400 µL PBS. The TRAP⁺-cells were quantified in 17 visual fields under the microscope (Olympus IX50).

To prepare the TRAP (tartrate resistant acid phosphatase) staining solution, 2 mg of phosphate disodium salt (N-5000, Sigma-Aldrich, St. Louis, MO, USA) was first dissolved in 200 μl of H₂O_dd. Acetate buffer was prepared by mixing 35.2 ml 0.2 M sodium acetate solution, 14.8 ml 0.2 M acetic acid solution and 50 ml H₂O_d. TRAP buffer pH 5.0 was then freshly prepared with 10 ml of acetate buffer, 2 ml of 0.3 M sodium tartrate, 200 μl naphthol AS-MX phosphate (10 mg/mL; N5000, Sigma-Aldrich, St. Louis, MO, USA), 20 μl Triton X‑100 (T8787, Sigma-Aldrich, St. Louis, MO, USA) and 7.78 ml H₂O_dd. TRAP buffer was preheated at 37 °C and then 0.3 mg/ml Fast Red Violet LB Stain (F-3381, Sigma-Aldrich, St. Louis, MO, USA) were dissolved in TRAP buffer. The TRAP staining solution was preheated at 37 °C until use. Cells were washed with prewarmed PBS (14190-094, Gibco™, Carlsbad, CA, USA) and fixed with 10% glutaraldehyde (G-5882, Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 15 min. After washing the cells twice with PBS, TRAP staining solution was added and incubated for 10 min at 37 °C. Then TRAP staining solution was removed and stained cells were washed with PBS. TRAP-positive cells (red) were counted using an Olympus IX50 microscope (Olympus, Shinjuku, Japan).

Femurs were fixed in 4% formaldehyde for 2 days, decalcified with 10% EDTA for 14 days, and embedded in paraffin. Sections (8 μm) were rehydrated using xylene (Sigma) and alcohol series. Hydrated slides were incubated in 0.2 M acetate buffer, pH = 5.0 (Sigma) for 1 to 1.5 hours at room temperature. Then the slides were transferred into prewarmed TRAP buffer, pH 5.0 (0.05 M acetate buffer, 0.03 M sodium tartrate, 0.1 mg/mL Naphtol AS‐MX phosphate, 0.1% Triton X‐100, 0.3 mg/mL Fast Red Violet LB stain (Sigma) and incubated for 30 minutes at 37°. Slides were washed with water and counterstained with 0.05% Fast Green (Sigma) for 45 seconds. After washing with water, the slides were dehydrated and mounted. Pictures of the distal trabecular area of the femur were taken with a 20× objective of an EVOS XL Core Imaging System (Thermo Fisher Scientific). Stitching was performed with Adobe Photoshop 21.1.0 (Adobe, San Jose, CA, USA), and quantification of TRAP staining was performed with bone histomorphometry software TrapHisto.⁽³⁰⁾