Anti dna histone h1

Neutrophils (3 × 10⁵) adhered to 0.001% poly-L-lysine-treated coverslips were stimulated with soluble LM (1 µg/mL, LM suspension group) or directly adhered to either LM- or polyLM-treated coverslips (50 µg/mL, polyLM group) and incubated at 37 °C. After 90 min, the neutrophils were fixed with 4% formaldehyde and blocked against nonspecific binding with 100% AB-positive human serum for 60 min. Cultures were stained with antibodies against pan-LM (1:50 dilution, Sigma), α1 LM chain (100 µg/mL, clone L9393 Sigma), α4 (100 µg/mL, 1:20 dilution, Santa Cruz, Santa Cruz, CA, USA), α5 (1:50 dilution, Millipore, Burlington, MA, USA), anti-human neutrophil elastase (1:500 dilution, Calbiochem) or anti-DNA/histone H1 (1:500 dilution, Millipore) for 1 h at room temperature. Then, goat anti-rabbit or anti-mouse secondary antibodies labeled with Alexa Fluor 488 or 546 (1:300 dilution, Thermo Scientific, Waltham, MA, USA) were added. The slides were mounted in ProLong Gold Antifade Mounting with DAPI (ThermoFisher). Images were obtained with a Zeiss DMi8 confocal microscope.

Neutrophils (10⁵/well) were seeded in culture dishes with poly-L-lysine-coated glass coverslips (Corning, USA), and incubated with 100 nM PMA or Leishmania promastigotes (1 parasite/1 neutrophil ratio) for 90 min at 35 ^oC with 5% CO₂ and fixed in 4% formaldehyde. Following, slides were stained with primary antibodies anti-Argonaute-2 and -Apoliprotein-AI (Abcam, USA), anti-elastase (Calbiochem, USA) or anti-DNA/histone H1 (Millipore, USA), followed by secondary antibodies goat-anti-rabbit or anti-mouse Alexa Fluor 488 or 546 (Molecular Probes, USA). The slides were mounted in ProLong Gold Antifade Mountant with DAPI (Thermo Fisher). Confocal images were taken in a Zeiss LSM 710.