Pa5 21721

Mouse bone specimens were first fixed and then decalcified using 10% EDTA (Sigma-Aldrich, St. Louis, MO, USA) for 14 days with constant shaking. For the histological assays, the detailed protocols were reported in a previous study.⁵⁸ In short, the samples were then dehydrated and embedded in optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA) or in paraffin. Four-μm-thick coronal-oriented femur sections were prepared for TRAP staining. Forty-μm-thick coronal-oriented femur sections were prepared for IF staining. The detailed protocols were described in a previous study.⁵⁸ Briefly, the sections were incubated with primary antibodies against mouse TLR2 (Santa Cruz Biotechnology, sc-21759, 1:200), Siglec15 (PA5-48221, Thermo Fisher Scientific, 1:100), ST3GAL1 (PA5-21721, Thermo Fisher Scientific, 1:50), and TRAP (Abcam, ab191406, 1:100) for 12 h at 4 °C. For sialic acid detection, biotinylated Maackia Amurensis Lectin II (MAL II) (Vector Laboratories, CA, USA) was used to label the α2,3 linkage, and biotinylated Sambucus Nigra Lectin (SNA) (Vector Laboratories, CA, USA) was used to label the α2,6 linkage. Fluorescein-conjugated streptavidin (Vector Laboratories, CA, USA) was used for the addition of a fluorescent label to biotinylated sialic acid conjugates. A Zeiss LSM 780 confocal microscope and an Olympus BX51 microscope were used for image capture.

The knee joint tissues of the mice were collected after upon killing and fixed in 10% buffered formalin phosphate for 48 hr. Samples were then decalcified in 10% ethylenediaminetetraacetic acid (EDTA, pH 7.4) for 2 weeks and embedded in paraffin or optimal cutting temperature (OCT) compound (Sakura Finetek). Four μm thick, sagittal-oriented sections of paraffin-embedded knee joints were processed for Tartrate-resistant acid phosphatase (TRAP) staining using a standard protocol (Sigma-Aldrich). Ten μm thick, sagittal-oriented sections of OCT-embedded knee joints were processed for immunofluorescence staining. The sections were incubated with primary antibodies against RANK (1:100, ab13918, Abcam), TLR2 (1:100, mAb12276, Cell Signaling), Maackia Amurensis Lectin (1:100, B-1265–1, Vector Laboratories), ST3GAL4 (1:100, 13546–1-AP, Proteintech), c-Fos (1:100, mAb2250, Cell Signaling), or ST3GAL1 (1:50, PA5-21721, Thermo Fisher Scientific) overnight at 4 °C. Then, the samples were incubated with secondary antibodies and DAPI (1:250, H-1200, Vector) in the dark for 1 hr at room temperature. The fluorescence images were taken by fluorescence microscopy (Olympus BX51, DP71) or confocal microscopy (Zeiss LSM 880) and analyzed by ImageJ software (National Institutes of Health, Bethesda).