Tnb buffer

Spleen was embedded in OCT compound (Sakura Finetechnical) and frozen in liquid N₂. The tissue segments were sectioned with a cryostat at 8 μm. Frozen sections were fixed in cold acetone and blocked in TNB buffer (PerkinElmer Life Science) containing 5% normal rat serum. To block endogenous biotin, the sections were further treated with the Streptavidin/Biotin Blocking Kit (Vector Laboratories), and endogenous peroxidase activity was quenched with 1% H₂O₂. The primary Abs were anti-CD19-FITC mAb (cat#553785, 1D3; BD Biosciences), anti-mouse dendritic cell marker-biotin mAb (cat#130-101-843, 33D1; Miltenyi Biotec), anti-CD11c mAb-biotin mAb (cat#553800, HL3; BD Biosciences) and anti-CD3ɛ-APC mAb (cat#100312, 145-2C11; Biolegend). The CD19 signal was amplified by incubation with Alexa Fluor 488-conjugated anti-rat IgG (cat#A11006; Life Technologies). The 33D1 and CD11c staining was revealed with a TSA signal amplification kit (PerkinElmer Life Science) according to the manufacturer's instructions. The sections were incubated with Streptavidin-HRP (PerkinElmer Life Science) followed by Tyramide-Cy3 conjugate. At the end of the staining, slides were washed and mounted with Vectashield (Vector Laboratories). The stained slides were examined with a BIOREVO fluorescence microscope (BZ-9000; KEYENCE).

Formalin-fixed paraffin-embedded decalcified tibia sections from 6-week-old and 8-week-old mice were obtained. For anti-Sp7 immunohistochemistry staining, antigen retrieval was performed using proteinase K (20 μg ml⁻¹) for 15 min. Endogenous peroxidases were quenched, and slides were blocked in TNB buffer (PerkinElmer), then stained with anti-Sp7 antibody (Abcam, ab22552) at a concentration of 1:200 for 1 h at room temperature. For activated caspase-3 IHCs, sections were stained with primary antibody (Cell Signaling Technology, 12692) at 1:500 overnight at 4 °C. Sections were washed, incubated with horseradish peroxidase (HRP)-coupled secondary antibodies, signals amplified using tyramide signal amplification, and HRP detection was performed using 3,3′-diaminobenzidine (DAB, Vector Laboratories) for 2–3 min. Slides were briefly counterstained with hematoxylin before mounting. Hematoxylin and eosin staining was performed using standard protocols. Quantification of Sp7-positive osteocytes and empty lacunae were performed in ImageJ imaging software in a blinded manner.

After 7-14 days of treatment, SCG cultures were gently fixed for 10-15 minutes in Cytofix/Cytoperm (BD, Cat. No. 554722), blocked in TNB buffer (Perkin Elmer, Cat. No. FP1020) for 1 hour at RT, stained with primary antibodies O/N at 4°C, stained with secondary antibodies for 1-2 hours at RT, followed by 5 minute DAPI stain and acquisition on Zeiss LSM 980 microscope with Airyscan2.