Biotinylated maackia amurensis lectin 2 malii

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.

ZIKV-infected cells in an 8-well chamber slide were fixed with 4% paraformaldehyde for 10 min followed by permeabilization with 0.25% Triton-X-100 in PBS. Cells were then blocked with 1% BSA in PBS for 1 h at room temperature. ZIKV envelope antigens were immunostained with primary anti-ZIKV envelope monoclonal antibody (EastCoast Bio) at 1:1000 dilution and secondary NL-493-conjugated anti-mouse IgG antibody (R&D Systems) for 1 h at room temperature. Cell nuclei were stained with Hoechst 33342 (Sigma) for 10 min. Immunofluorescence was detected with a fluorescent microscope (Nikon).
For sialic acid staining, cells were fixed with 4% paraformaldehyde for 10 min. Biotinylated elderberry bark lectin (SNA, Vector labs), biotinylated Maackia amurensis lectin II (MAL-II, Vector labs) and FITC-conjugated wheat germ agglutinin lectin (WGA, Sigma) were used to stain α2,6-linked, α2,3-linked and total sialic acid, respectively. A final concentration of 20 µg/ml of the lectins was added into fixed cells for 1 h at room temperature. Streptavidin NL-493 (R&D systems) were added at 1:1000 dilution for 1 h at room temperature. The nuclei were stained with DAPI (Sigma) and the fluorescence images were captured using Cytation 5 imager (BioTek).