Ab90812

The paraffin sections of right lung lobes were deparaffinized. After inhibiting endogenous peroxidase activity and repairing antigen, the non-specific binding sites were blocked with 10% bovine serum albumin (BSA) and incubated with primary antibodies including rabbit anti-NE (bs-23549R, Bioss, Beijing, China) and rabbit anti-CitH3 (AF0863, Affinity, Colorado, United States) for 12 h at 4°C and washed with PBST, then staining tissues with Goat Anti-Rabbit IgG H&L (Alexa Fluor®488, ab150077, Abcam, London, UK) and Goat Anti-Rabbit IgG H&L (Alexa Fluor®647, ab150115, Abcam) secondary antibodies, respectively. The expression levels of NE and CitH3 were analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, United States) after being detected by an optical microscope (Vectra 3, PerkinElmer, Waltham, United States) (Liu et al., 2022 (link)).
In addition, dewaxed and blocked tissues were incubated with FITC anti-mouse F4/80 antibody (1:50, 123107, Biolegend, California, United States), PE anti-mouse Ly-6G antibody (1:50, 127607, Biolegend) and FITC anti-mouse MPO antibody (1:50, ab90812, Abcam) for 1 h at 37°C, respectively, followed by staining with DAPI (1:200) to label cell nuclei for 10 min at 37°C. The expressions of these genes were detected by optical microscope and quantified using ImageJ software (National Institutes of Health).

Mice were euthanized at 24 hours after surgery for IF staining, which was conducted as previously described (3 (link), 19 (link)). Brain sections with 8-μm thickness were incubated with 5% bovine serum albumin (BSA) and 0.3% Triton X-100 for 2 h at room temperature. Brain sections were incubated overnight at 4°C with primary antibodies: anti-CD68 antibody (Abcam, ab237968), anti-CD16 antibody (Invitrogen, MA1-7633), anti-CD86 antibody (Invitrogen, MA1-10299), anti-CitH3 antibody (Abcam, ab5103), anti-MPO antibody (Abcam, ab90812), anti-NE antibody (Abcam, ab68672). Sections were incubated with secondary antibodies for 2 h at room temperature. Fluoro-Jade C (FJC) staining was performed to detect neuronal damage according to the manufacturer’s protocol (Roche Inc., Basel, Switzerland). The sections were visualized using a fluorescence microscope (Leica, Germany). ImageJ software was used to analyze the results.

The toxic effects of inhibitors were initially determined to confirm that the indicated concentrations of these inhibitors would not cause obvious cell death. The isolated neutrophils were seeded in 24-well plates at a density of 10⁵ cells/well and then pre-treated with the inhibitors [1 μM Ro 31-8220 (Sigma, R136), 5 μM DPI (Sigma, D2926), 100 μM ABAH (Sigma, A41909), 10 μM cytochalasin B (Sigma, C2743) or 10 μM MMP8 inhibitor I (Calbiochem, 444237)] for 30 min before stimulation with 100 nM PMA or S. suis (MOI = 2). Cells without stimulation served as a control. NET formation was visualized by staining with a 100 nM solution of the extracellular nucleic acid dye SYTOX Green (Invitrogen, S11368) for 10 min and then measured in a BioTek synergy HT plate reader at excitation/emission wavelengths of 485/530 nm. Cells were incubated with SYTOX Orange for 10 min to further investigate NET formation. After washing, cells were further incubated with Ms mAb against myeloperoxidase (FITC) (Abcam, ab90812) for 1 h. After five washes, all slides were mounted with 50% glycerol and covered with glass cover slips, followed by an analysis with an LSM 880 confocal microscope (ZEISS) and ZEN 2.3 LITE software (ZEISS). Wavelengths of 488 nm and 561 nm were used because the excitation/emission wavelengths of FITC and SYTOX Orange are 488/520 and 547/570 nm, respectively.

Brains were removed and fixed with 4% paraformaldehyde at 4 °C overnight. Frozen sectioning was performed after sucrose gradient dehydration followed by blocking with 10% goat serum containing 0.5% Triton X-100 at room temperature for 30 min. Brain sections were incubated with different primary antibodies at 4 °C overnight, including anti-MPO (Abcam, ab90812, 1: 1000), anti-NeuN (Abcam, ab177487, 1: 1000), anti-CD31 (Abcam, ab222783, 1: 1000), anti-Iba-1 (Abcam, ab153696, 1: 1000), anti-GFAP (Abcam, ab7260, 1: 1000) antibodies. The sections were washed with PBS 3 times and incubated with the mixture of Alexa Fluor 488 conjugated goat-anti-mouse IgG (Beyotime, A0428, 1: 500) or Alexa Fluor 555 conjugated donkey-anti-rabbit IgG (Beyotime, A0453, 1: 500) for 1 h in darkness at room temperature, followed by staining with DAPI for 5 min. After being washed with PBS 3 times, sections were mounted with fluorescent mounting medium (Dako, S3023), and examined by a confocal laser scanning microscope (LSM 780, Carl Zeiss).

Anti-mouse Ly6G (IA8; 127608; BioLegend, CA, USA), anti-mouse CD11b (M1/70; 101212; BioLegend), anti-mouse CD63 (NVG-2; 143920; BioLegend), anti-mouse CXCR2 (SA044G4; 149315; BioLegend), anti-mouse CXCR4 (L276F12; 146511; BioLegend), and anti-mouse CD4 (GK1.5; 100406; BioLegend) antibodies were used to stain the surface molecules of samples. For intracellular staining, anti-mouse IFN-γ (XMG1.2; 505809; BioLegend) and anti-mouse IL-17A (TC11-18H10.1; 506903; BioLegend) antibodies were used. For NET components staining, anti-mouse MPO (ab90812; Abcam, MA, USA), anti-mouse CitH3 (ab5103; Abcam), anti-mouse PAD4 (ab96758; Abcam), anti-mouse NE (ab38672; Abcam), anti-mouse β-actin (8457S; Cell Signaling Technology, MA, USA), Alexa Fluor 488 goat anti-rabbit IgG (ab150077; Abcam), Alexa Fluor 555 goat anti-rabbit IgG (ab150078; Abcam), and Alexa Fluor 647 donkey anti-rabbit IgG (101,231; BioLegend) antibodies were used.