Immunofluorescence Analysis

To detect the citrullination of histone H3 in mouse neutrophils or dHL60, 2.5 × 10⁵ cells were seeded in a 35-mm poly-_L-lysine-coated glass bottom dish (MATSUNAMI) and stimulated with PMA, PMA + DDS, or ICs. The cells were then fixed with 4% paraformaldehyde for 10 min at room temperature and incubated in HBSS supplemented with 10% normal goat serum (Sigma), bovine serum albumin (Sigma) and 0.01% Tween 20 for 1 h for blocking. The cells were incubated first with hamster anti-histone H3 (citrulline R2 + R8 + R17) (citH3) antibody (11-11B-4F) (Yotsumoto et al., 2017) and then with anti-hamster IgG antibody coupled with Alexa Fluor 488 or 647 (Thermo Fisher Scientific). DNA was labeled using DAPI (DOJINDO).

To detect the citrullination of histone H3 in lungs, C57BL/6J mice were administrated PBS or LPS (50 μL at a concentration of 0.25 mg/ml) intranasally. After 24 h, the lungs were harvested and embedded in SCEM compound (SECTION-LAB). The cut surface was covered with adhesive film (Cryofilm type IIC9, SECTION-LAB, Japan) and frozen sections (5 μm) were prepared with a microtome (CM3050S, Leica Microsystems). The resulting sections were post-fixed with 100% EtOH for 10 s and 4% PFA/PBS(-) for 10 s, rinsed with PBS(-) for 20 s, and incubated with TN Blocking Buffer [0.1 M Trizma Base, pH 7.5, 0.15 M NaCl, 0.5% (w/v) blocking reagent (PerkinElmer, FP1020)] for 1 h at room temperature. The sections were then incubated with anti-citH3 antibody (Abcam, #ab5103, 1:250) or Human/Mouse MPO Antibody (R&D Systems, AF3667, 1:100) in TN blocking buffer for 1 h at room temperature. After three washes with PBS(-), the sections were incubated with Cy3 donkey anti-rabbit IgG (1:1000, Biolegend) or AlexaFluor488 donkey anti-goat IgG (1:1000, Jackson ImmunoReserch) in TN blocking buffer for 1hr in the dark at room temperature. After three washes with PBS(-), the sections were counterstained with DAPI, and the slides were mounted onto cover slips using mounting media (FluorSave Reagent, 345789, Merck Millipore).