Leucocyte acid phosphatase kit

Hind paws were prepared, fixed in formalin for 24 hours and bones decalcified. Serial paraffin sections were stained with haematoxylin/eosin (H&E), toluidine blue (TB) or were stained for tartrate‐resistant acid phosphatase (TRAP) expression using leucocyte acid phosphatase kit (Sigma‐Aldrich). Histomorphometrical analysis was accomplished using the Osteomeasure software (Osteomeasure 7, Osteometrics, Georgia, USA).

Bone marrow-derived cells were freshly isolated from femur and tibia by flushing the marrow using α-MEM supplemented with 1% Penicillin/Streptomycin and 10% FCS (Life Technologies). Cells were seeded in 24-well plates at a density of 250 000 cells/well and their proliferation was stimulated with 25 ng/ml macrophage colony-stimulating factor (M-CSF, R&D Biosystems) for the rest of the experiment. After 3 days, differentiation was stimulated by supplementing culture media with 50 ng/ml receptor activator of nuclear factor kappa-Β ligand (RANKL, R&D Biosystems). Cells were stimulated with RANKL and M-CSF every second day and after 10 days post-seeding, cells were fixed with 4% PFA and stained for TRAP using Leucocyte acid phosphatase kit (Sigma Aldrich), according to manufacturer’s protocol. Multinucleated TRAP positive cells (nuclei number ≥3) were imaged with Leica DMI 6000B microscope and their number and total area were quantified using the OsteoMeasure software (OsteoMetrics, Decatur).
To study the impact of MEK (U0126) or PI3K (LY294002) inhibition on osteoclast differentiation, U0126 (Selleckchem) were added to a final concentration of 5 µM and LY294002 2.5 µM, respectively, at various time points of differentiation and its effects on osteoclast differentiation were monitored by quantification of osteoclast number and area from images of TRAP staining, as described above.

Osteoclasts were identified as TRAcP⁺ multinucleated cells by using a commercially available Leucocyte acid phosphatase kit (Sigma–Aldrich). The staining procedure was performed following the manufacturer's instructions. Nuclei were counterstained by 4′6‐diamidino‐2‐phenylindole (DAPI). The TRAcP⁺ multinucleated cells were categorized as cells containing 3–10, and >10 nuclei, and the number of cells in each category was counted using a combination of light and fluorescence microscopy (Leica DFC320; Leica Microsystems, Wetzlar, Germany).

Osteoclasts were identified by histochemical staining of tartrat-resistant acid phosphatase (TRAP) (Leucocyte Acid Phosphatase Kit, Sigma Aldrich Chemie GmbH, Steinheim, Germany) and counted in the whole peripheral callus. Additional paraffin sections were used for immunostaining of sphingomyelin phosphodiesterase 3 (Smpd3) by the use of a polyclonal anti-mouse Smpd3 antibody (Santa Cruz Biotechnology Inc., Heidelberg, Germany). To detect the primary antibody, labeled-streptavidin-biotin method (LAB-SA, Histostain-Plus Kit, Life Technologies GmbH, Darmstadt, Germany) was used and 3-amino-9-ethylcarbazole (AEC) was used as chromogen (Zytomed Systems, Berlin, Germany). The monoclonal non-phospho (active) β-catenin antibody (Cell Signaling, Danvers, MA, USA) and the polyclonal anti-collagen type II antibody (Rockland, Gilbertsville, PA, USA) were used together with the Vectastain ABC kit (Vector Laboratories, Burlingham, CA, USA) to detect the stabilized active form of endogenous β-catenin and collagen type II, respectively. Finally, counterstaining with hematoxylin (Waldeck, Münster, Germany) was performed. The sections were qualitatively evaluated under light microscopy.

Histology was performed as previously described [29 (link)]. Overall cellular and tissue structure was visualized with haematoxylin–eosin (H&E) staining. Glycosaminoglycans were visualized by staining with 1% Alcian blue 8-GX (Sigma-Aldrich) and Nuclear Fast Red (Sigma-Aldrich). Calcium deposits were visualized with 2% Alizarin Red S (Sigma-Aldrich). Osteoclasts were stained with tartrate resistant acid phosphatase (TRAcP; Leucocyte acid phosphatase kit, Sigma-Aldrich).

To assess the formation of osteoclasts, tartrate‐resistant acid phosphatase (TRAP) activity was measured via a leucocyte acid phosphatase kit (Sigma‐Aldrich). Cells were incubated in the presence of FHA and HA in medium environment containing RANKL for 6 days. Before staining, cells were washed from the materials and cultured in culture plates. Cells were then washed with PBS for three times and fixed utilizing 4% paraformaldehyde for 15 minutes. Washing with PBS three times again was followed. The experiment was performed according to the protocol. The number of osteoclasts per disc was quantified and averaged. F‐actin staining was then performed to investigate FHA effect on F‐actin ring formation as previously reported. Mature osteoclasts were seeded on glass coverslips. Cells were then fixed with 4% formaldehyde in PBS for ten minutes and permeabilized using ice‐cold acetone. Coverslips were subsequently incubated with rhodamine phalloidin stock solution (Cytoskeleton, Denver, CO, USA) for 20 minutes and observed using a confocal imaging system.

Bone marrow monocytes were cultured in 96‐well plates in full medium containing M‐CSF and allowed to adhere overnight. The medium was replaced and the cells were treated with 30 ng/mL M‐CSF and 50 ng/mL RANKL for additional 5 days. The medium was replaced every 2 days. TRAP staining was then performed with a leucocyte acid phosphatase kit (Sigma) according to the manufacturer’s instructions. TRAP‐positive multinucleated cells (≥3 nuclei) were scored as osteoclasts.