Tyramide signal amplification system

The mice were anesthetized using pentobarbital sodium (50 mg/kg, intraperitoneally) on day 0, day 1, day 2, day 4, or day 7 after surgery. Femurs were removed, fixed in 4% paraformaldehyde, demineralized in 22.5% formic acid and 340 mM sodium citrate solution for 24 h, and embedded in paraffin. Immunostaining was performed as previously described (Okada et al., 2016 ). Briefly, the sections were incubated with anti-ALP antibody at a dilution of 1:100, anti-Osterix antibody at a dilution of 1:200, or anti-SDF-1 antibody at a dilution of 1:200 followed by incubation with the appropriate horseradish peroxidase-conjugated secondary antibody. Positive signals were visualized using the tyramide signal amplification system (PerkinElmer, Waltham, MS, USA), and sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and photographed using a fluorescence microscope (E800; Canon, Tokyo, Japan) with a CCD camera or a confocal microscope (C2 Si; Nikon, Tokyo, Japan).

Lung cryosections were stained with anti-CD3-Cy3 (17A2), anti-B220-AlexaFluor647 (RA3-GB2) (all prepared in-house) and anti-CD21/35-FITC (7G6; BD) antibodies. For chemokine staining, goat anti-CXCL12 (polyclonal, PeproTech) and goat anti-CXCL13 (R&D Systems) antibodies were detected with donkey anti-goat HRP (Jackson ImmunoResearch) and amplified with a Tyramide Signal Amplification system (PerkinElmer). Imaging was performed on an epifluorescence microscope equipped with an F-View II camera (Olympus). The numbers of BALT structures per section were counted using the polygon tool in cellSens software (Olympus).

PP tissues were fixed with zinc formalin fixative (Polysciences) and embedded into paraffin. Paraffin sections of PPs were deparaffinized and treated with 0.3% H₂O₂ in PBS for 20 min at room temperature to quench an endogenous peroxidase activity and then sections were boiled with citrate buffer, pH 6.0, for 20 min to antigen retrieval of both IL-22BP and pSTAT3. The sections were incubated with 0.5% blocking buffer (PerkinElmer) in PBS for 30 min at room temperature and then with primary antibodies overnight at 4°C. The binding of anti–IL-22BP (AF1087; R&D Systems) and anti–phospho-STAT3 (Tyr705; Cell Signaling Technology) was followed by biotinylated secondary antibodies and visualized by a Tyramide Signal Amplification system (PerkinElmer). The sections were analyzed with an SP5 confocal laser microscope (Leica Microsystems).