Fitc conjugated anti ifn γ antibody

Lymphocytes from spleens were resuspended in staining buffer (PBS containing 3% of fetal bovine serum) and stained for 30 minutes at 4°C with an APC/Cy7-conjugated anti-mouse CD3 antibody (Lot. 100330, BioLegend), PE/Cy7-conjugated anti-mouse NK-1.1 antibody (Lot. 108714, BioLegend), APC-conjugated anti-mouse CD4 antibody (Lot. 100412, BioLegend), PerCP-conjugated anti-mouse CD8a antibody (Lot. 100732, BioLegend) as well as their corresponding isotype control antibodies as a negative control. Cells were fixed and permeabilized using the fix/perm kit (Lot. 88-8823-88, eBioscience). Intracellular IFN-γ and IL-4 staining was performed on the cells stained with a PE-conjugated anti-IL-4 antibody (Lot. 5014104, BioLegend) and FITC-conjugated anti-IFN-γ antibody (Lot. 505806, BioLegend) and its isotype control antibodies as a negative control. Flow cytometry was performed on a BD Canto II flow cytometer (BD Biosciences).

Skeletons were collected from mice and fixed in 10% neutral-buffered formalin for 4 days at 4°C, followed by decalcification with 14% ethylenediaminetetraacetic acid (EDTA) for 2 weeks. Tissue sections were stained with hematoxylin and eosin (H&E) according to standard protocols. Anti-osteocalcin monoclonal antibody (clone #E-6) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Safranin O (#S2255) and fast green FCF (#F7262) were purchased from Sigma-Aldrich (Darmstadt, Germany). For immunofluorescence experiments, preparations were incubated for 1 h at room temperature with Alexa Fluor® 488- or PE-conjugated anti-mouse CD3 antibody (clone #17A2, BioLegend, San Diego, CA, USA), APC-conjugated anti-TNF-α antibody (clone #MP6-XT22, BioLegend), FITC-conjugated anti-IL-17A antibody (clone #REA660, Miltenyi Biotec, Bergisch Gladbach, Germany), and FITC-conjugated anti-IFN-γ antibody (clone #XMG1.2, BioLegend). Homemade anti-DUSP22 monoclonal antibody (clone #C8), recognizing both human and mouse DUSP22 proteins, was generated by immunization of mice with peptides (murine DUSP22 epitope: ¹⁷⁰GKYKEQGRTEPQPGARRWSS¹⁸⁹). 4′,6-diamidino-2-phenylindole (DAPI) was used for counterstaining (Merck, Darmstadt, Germany). Fluorescence was detected using a fluorescence microscope (Leica TCS SP5II).