Rabbit anti cith3 antibodies

The above treated neutrophils were fixed by 4% paraformaldehyde in PBS overnight at 4 °C, then permeabilized with 0.1% TritonX-100 in PBS for 20 min, and washed three times with PBS. Cells were blocked with 1% BSA, 0.1% Tween 20 in PBS for 2 h at room temperature, and then incubated with rabbit anti-MPO antibodies (Abcam, UK, 1:200 diluted in 0.1% Tween 20 in PBS) overnight at 4 °C. Next, neutrophils were gently washed three times with PBS and incubated in the dark with goat anti-rabbit IgG antibody (Alex Flour 647 nm, 1:500 diluted in the PBS) for 2 h at room temperature. Before staining with 100 ng/ml DAPI, neutrophils were incubated with rabbit anti-CitH3 antibodies (Abcam, UK, 1:200 diluted in 0.1% Tween 20 in PBS) overnight at 4 °C, then washed three times and incubated with Alexa fluor 488 nm goat anti-rabbit IgG fluorescent secondary antibody, finally washed with PBS and ProLong Diamond Antifade Mountant (Thermo, USA) was added to each coverslip, the coverslips were placed on the slides, and then observed with a confocal fluoresce microscope (FV3000 Olympus, Japan). Images were analyzed with the ImageJ software from National Institutes of Health^{33 ,61 (link)}.

The skin and ankle joints were fixed in neutralized 10% formalin, embedded in paraffin, and sliced. For immunohistological analysis using AMC antibodies, we prepared modification buffer by mixing Reagent A (20% H₂SO₄, 25% H₃PO₄, and 0.025%FeCl₃) and Reagent B (1% diacetyl monoxime, 0.5% antipyrine, 1 M acetic acid) at a 2:1 ratio (volume/volume). The sections were covered with the modification buffer and incubated in a light-proof container at 37 °C for 2.5 h in order to modify citrulline residues. Then, the sections were incubated with rabbit AMC polyclonal antibodies diluted 1:3200 in 2% BSA in PBS to detect modified citrulline residues. Then, the sections were incubated with HRP-conjugated goat anti-rabbit IgG (H + L) (Bio-Rad, Hercules, CA, USA) for 30 min at room temperature. The sections were also stained with DAB (Nichirei Biosciences, Tokyo, Japan) and hematoxylin. To detect citrullinated histone 3 (CitH3), the sections were incubated with rabbit anti-CitH3 antibodies (Abcam, Cambridge, MA, USA) diluted 1:200 as primary antibodies. HRP-conjugated goat anti-rabbit immunoglobulin (IgG) (H + L) (Bio-Rad) diluted 1:1000 was used as a secondary antibody. The sections were also stained with DAB (Nichirei Biosciences) and hematoxylin.