Alexa fluor 568 647 conjugated secondary antibodies

The levels of cf-DNA and MPO-DNA complexes were measured using MPO ELISA kit (Finetest, China) and dsDNA Assay Kit (Invitrogen, USA) as previously described. Mouse pancreas was freshly collected, fixed with 10% neutral buffered formalin and embedded in paraffin. Pancreatic tissues were sectioned into 3 μm. After antigen repair with citric acid-containing antigen repair buffer under high pressure, pancreatic tissue sections were blocked with 5% (wt/vol) BSA and incubated with the primary antibody Histone H3 (citrulline R2+R8+R17, ab5103, 1:1000, Abcam, USA) and Myeloperoxidase (GB12224, 1:1000, Servicebio, China) overnight at 4°C. After three washes, the sections were subsequently incubated with Alexa Fluor 568/647 conjugated secondary antibodies (1:1000, Invitrogen, USA) for 1 h at room temperature and counterstained with DAPI (1:2000, Sigma, Sweden).

Purified neutrophils were seeded onto coverslips coated with 0.001% poly-L-lysine (Beyotime, China). Neutrophils were fixed in 4% (vol/vol) PFA for 30 min at room temperature, washed with PBS and permeabilized with 0.1% Triton X-100 for 10 min at 4°C. After blocking with 3% (wt/vol) BSA for 90 min at 37°C, neutrophils were incubated overnight at 4°C with primary antibody Histone H3 (citrulline R2+R8+R17, ab5103, 1:1000, Abcam, USA) and Myeloperoxidase (ab25989, 1:1000, Abcam, USA). Neutrophils were subsequently incubated with Alexa Fluor 568/647 conjugated secondary antibodies (1:1000, Invitrogen, USA) for 1 h at room temperature followed by DAPI solution for 10 min. NETs formation was visualized using a confocal microscope (Carl Zeiss, Jena, Germany).

Antigen repair and blocking were performed on the Pancreatic tissue sections as previously described. Then the sections were incubated with the primary antibody PAD4 (ab214801, 1:500, Abcam, USA) and Ly6G (BE0075-1, 1:200, InVivoMAb, USA) overnight at 4°C. After three washes, the sections were subsequently incubated with Alexa Fluor 568/647 conjugated secondary antibodies (1:1000, Invitrogen, USA) for 1 h at room temperature and counterstained with DAPI (1:2000, Sigma, Sweden).