B 1265 1

Staining procedures were similar as previously mentioned for LTL stain, but antigen retrieval was attained by using PROTEASE 1 for 4 minutes. Slides were incubated with biotinylated Maackia Amurensis (MAL II) lectin at room temperature for 1 hour (dilution 1:100 in PBS, Vector laboratories, catalog no. B-1265-1, Burlingame, CA). The lectin is obtained from their seeds, which stains with NeuAc in an α-2,3 linkage.

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).