Goat anti rabbit igg h l alexa fluor 568

Four days after inoculation SCN-infested root segments were fixed in glutaraldehyde and paraformaldehyde and processed as described in the Supplemental Experimental Procedures. After overnight incubation with anti-AAT_Rhg1 roots were treated with secondary antibody Alexa Fluor 568 goat anti-rabbit IgG H&L (Abcam ab175471) for 2 h and then imaged right away by confocal microscopy.

100 µL/well of freshly isolated PMN in RPMI-1640 medium at a concentration of 2 million/mL were seeded in Nunc™ Lab-Tek™ Permanox chamberslides (Thermo Fisher Scientific). The chamber slides were incubated with 50 pg/cell monosodium urate (MSU) crystals for 4 h at 37 °C to allow NET formation. The medium alone served as unstimulated control. Afterwards, samples were fixed using 2% paraformaldehyde for 20 min at RT. After washing of the samples three times with PBS, NETs/PMN were permeabilized using 0.1% Triton X-100 in PBS for 5 min at RT before blocking with 10% FCS, 2% BSA in PBS for 1 h at RT. Mouse anti-DNA-IgM (CBL186, Merck KGaA; diluted 1:100) and rabbit anti-human MPO antibody (1:100, ab9535, Abcam, Cambridge, UK, poly-clonal IgG) were incubated in blocking buffer ON at +4 °C. After washing three times with PBS, Alexa Fluor™ 568 goat anti-rabbit IgG (H+L) (1:400, ab175471, Abcam, Cambridge, UK, polyclonal IgG) and Cy™5 AffiniPure goat anti-mouse IgM (H+L) (1:400, 115-175-075, Jackson ImmunoResearch Europe Ltd.) in blocking buffer were used as a secondary antibody. The secondary antibodies were incubated with the DNA stains DAPI (ThermoFisher) and Sytox^TM Green (ThermoFisher) for 1 h at RT. After another washing step with two times PBS and one-time deionized water, the chambers were removed and slides were mounted using DAKO fluorescent mounting medium (Agilent).