Naphthol as mx phosphate

Differentiated cells were washed with PBS and treated with 4% paraformaldehyde solution (Fujifilm Wako) for 10 min at room temperature. The cells were washed again with PBS and then stained with a TRAP staining solution containing 50 mM sodium tartrate, 45 mM sodium acetate, pH 5.2, 0.1 mg/mL naphthol AS-MX phosphate (Sigma-Aldrich, St. Louis, MO, USA), and 0.6 mg/mL fast red violet LB (Sigma-Aldrich) at pH 5.2 for 1 h or longer at room temperature. The stained cells were observed under the EVOS XL Core microscope (Thermo Fisher Scientific), and TRAP-positive cells that stained red and contained three or more nuclei were counted.

Non-skeletal organs were histologically analysed in the German Mouse Clinic as
described60 (link). All organs were fixed in 4% buffered formalin before
being embedded into paraffin. Sections of 2 μm thickness were stained
with haematoxylin and
eosin according to standard
protocols. For bone-specific analyses, dissected skeletons were fixed in 3.7%
PBS-buffered formaldehyde for
18 h and then transferred into 80% ethanol. Vertebral bodies L1 to L4 were dehydrated in ascending
alcohol concentrations, before they were embedded into methylmethacrylate. Sections of
4 μm thickness were cut in the sagittal plane using a Microtec rotation
microtome. These were stained by von Kossa/van Gieson (for static hisotmorphometry)
and toluidine blue (for cellular histomorphometry) staining procedures as
described62 (link). To determine the bone formation rate, all mice were
injected twice with calcein 9 and
2 days before being killed. Static, cellular and dynamic histomorphometry at
trabecular bone surfaces was carried out according to the guidelines of the American
Society for Bone and Mineral Research63 (link) using an OsteoMeasure system (Osteometrics
Inc., USA). TRAP
activity staining was performed on decalcified sections using Naphthol AS-MX phosphate (Sigma) and Fast Red Violet
LB salt (Sigma) in 40 mM acetate buffer (pH
5).

Cells were washed with phosphate buffer saline, fixed with 10% neutral buffered formalin, and stained for 30 min using 120 mM Tris buffer (Sigma-Aldrich) containing 1.8 mM fast red TR (Sigma-Aldrich) and 0.9 mM naphthol AS-MX phosphate (Sigma-Aldrich) at 37°C.

Osteoclast differentiation was assessed by TRAP staining as described previously44 (link). Briefly, cultured cells were fixed with 10% (v/v) formalin in PBS followed by ethanol/acetone (1/1, v/v), and stained for TRAP with 0.1 mg/ml naphthol AS-MX phosphate (Sigma) and 0.6 mg/ml fast red violet LB salt (Sigma) in 0.1 M sodium acetate buffer, pH 5.0, containing 50 mM sodium tartrate. The TRAP-positive cells were photographed with a BZ-8000 digital microscope (Keyence), and counted manually. In some experiments, TRAP activity was quantified as follows. Cells in 96-well plates were fixed and incubated for 5 min in 0.1 ml of 10 mM p-nitrophenyl phosphate (Sigma) in 0.1 M sodium acetate buffer, pH 5.0, containing 50 mM sodium tartrate. After incubation, the solution was transferred to a tube containing 0.1 ml of 0.1 N NaOH, and the absorbance at 405 nm was determined.

Before immunohistochemical staining, spleen sections were fixed in ice-cold acetone for 10 min. Endogenous peroxidase activity was blocked by 10 min room temperature incubation in 0.3% H₂O₂ before staining. Sections were stained in 300-fold diluted biotinylated PNA (B-1075, Vector Laboratories) for 1 h in a humidified chamber, followed by Streptavidin-alkaline phosphatase (AP) (7100-04, SouthernBiotech). Bound AP was then visualized by enzymatic detection with 0.125 mg ml⁻¹ of naphthol-AS-MX phosphate (855-20 ML, Sigma), 0.25 mg ml⁻¹ Fast Blue BB salt (44670, Sigma) and 2 mM levamisole (1359302, Sigma) in 0.1 M Tris-HCl (pH 8.5)8 (link). Ten min later, stained sections were washed in PBS and mounted in 30% glycerol.