2 4 amidinophenyl 1h indole 6 carboxamidine dapi

NFAT3 localization in NMCs was analyzed using confocal microscopy. Briefly, NMCs were isolated and cultured on laminin pre-coated Lab-Tek chamber slides (Thermo Fisher Scientific, Inc.). Following treatment, cells (1×10⁵ cells/well) were fixed with 4% paraformaldehyde at room temperature for 30 min, permeabilized using 0.2% Triton X-100 and washed with PBS. Subsequently, cells were incubated in 1 ml blocking reagent (SuperBlock Blocking Buffer; Thermo Scientific™; Thermo Fisher Scientific, Inc.) with primary rabbit anti-NFAT3 (1:200; cat. no. sc-8405; Santa Cruz Biotechnology, Inc.) and primary mouse anti-α-actinin antibodies (1:200; cat. no. A2543; Sigma-Aldrich; Merck KGaA) in a cold room at 4°C for 24 h. Following extensive washing, NMCs were incubated with Alexa Fluor 594-labeled goat anti-rabbit (1:1,000; cat. no. Z25307; Invitrogen; Thermo Fisher Scientific, Inc.) and Alexa Fluor 488-labeled goat anti-mouse antibodies (1:1,000; cat. no. A20181; Invitrogen; Thermo Fisher Scientific, Inc.) at 4°C for 30 min. Finally, cells were mounted with mounting media containing 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI, 1:1,000) (Vector Laboratories, Inc.; Maravai LifeSciences). A Carl Zeiss 710 confocal microscope (Carl Zeiss AG; magnification, ×40) was used to image the NFAT3, α-actinin and DAPI staining.

The formalin-fixed and paraffin-embedded sections were deparaffinized and incubated with Histo VT One (Nacalai Tesque, Kyoto, Japan) for antigen retrieval according to the manufacturer’s instructions. After cooling, an anti-IL-38 antibody (Abcam, Cambridge, UK) was applied and incubated overnight at 4°C. DyLight 549-labeled anti-rabbit IgG antibody (Vector Laboratories, Burlingame, CA) was used as a secondary antibody. For double-staining procedures, anti-IL-38, anti-CD3 (Leica Biosystems, Buffalo Grove, IL), anti-CD19 (Santa Cruz Biotechnology, Dallas, TX), anti-CD68 (DAKO, Santa Clara, CA), and anti-myeloperoxidase (MPO) antibodies (R&D systems, Minneapolis, MN) were applied and incubated overnight at 4°C. Then, the slides were incubated with secondary antibodies conjugated to fluorescent dyes for 1 h at room temperature, visualizing the nuclei with 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI) (Vector Laboratories). Images were obtained with a confocal microscope TCS SP8 X (Leica Microsystems, Wetzlar, Germany). The antibodies used are listed in Supplemental Table 1*.