Bz x810 microscopy

Tissue samples from the mice were fixed with 4% paraformaldehyde for 24 h, decalcified in 12% EDTA for 10 d, embedded in paraffin, sectioned, deparaffinized and stained with hematoxylin and eosin (H&E) and Alcian blue. For IHC staining, antigen retrieval was performed via incubation with Liberate Antibody Binding Solution (Polysciences, USA) for 20 min at RT. Non-specific antigens were blocked using Blocking One (Nacalai Tesque) for 1 h at RT. For antibodies generated from the mouse host, tissues were blocked with ReadyProbes™ Mouse on Mouse IgG Blocking Solution (Invitrogen) for 60 min at RT. Antibodies (diluted in Can Get Signal Immunostain Solution B, TOYOBO) were incubated overnight at 4℃. After rinsing in PBST buffer, samples were incubated with Alexa Fluor secondary antibodies diluted in Can Get Signal Immunostain Solution B for 1 h at RT. A mounting medium with DAPI (Vector Laboratories) was used to counterstain the nuclei. Images were captured using a BZ-X810 microscopy (Keyence, Japan). The antibodies for IHC are listed in Additional file 1: Table S3.

Osteoclast differentiation was performed as previously described (7 (link)). Briefly, bone marrow cells isolated from male femurs were cultured in α-MEM supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS) in the presence of murine Macrophage colony-stimulating factor (M-CSF) (30 ng/ml). After 3 days, adherent cells were treated with M-CSF (30 ng/ml) and murine Receptor activator of nuclear factor kappa beta ligand (RANKL) (10 ng/ml). The medium was changed every other day. After 3 days, cells were stained by tartrate-resistant acid phosphatase (TRAP) staining (Millipore Sigma) and images were captured using the BZ-X810 microscopy (Keyence) using 10× objective lens. Cells were treated and collected according to each set of experiments. TRAP + cells containing three or more nuclei/cell were quantified. Total mature osteoclasts were counted and represented as TRAP + cells/well.

BALB/c female mice (n = 3-5/group) were injected in the right gastrocnemius muscle with 2D216 (200 nmol/mouse) with or without MPLA (10 ng/mouse). AS01B was used as a control. After 2 and 24 h, mice were bled and sera were assessed for cytokine (TNF-α and IL-6) and C-reactive protein (CRP) levels by Luminex multiplex cytokine assay (Thermo Fischer Scientific) and ELISA. After 24 h, the right gastrocnemius muscles were harvested and histological sections with 5 μm thickness were stained with hematoxylin and eosin (H&E) and analyzed by BZ-X810 microscopy (KEYENCE, Osaka, Japan).