Myeloperoxidase

Microscope slides were cleaned with isopropyl alcohol and coated with 0.01 mg/ml of poly-L-lysine at 4°C overnight. Then, 2.2 × 10⁵ neutrophils in 80 μl RPMI-1640 medium were seeded on the slides and incubated for 30 min at 37°C, 5% CO₂ for cell attaching. Whole C. albicans cells or selected stimulants including isolated glucans, mannans, isolated mixture of cell wall proteins (CWP) or purified proteins (enolase, Als3, Saps) were then added in the volume of 20 μl of RPMI at various range of concentrations. Neutrophils, treated for 3 h at 37°C, 5% CO₂ with 25 nM PMA (Sigma-Aldrich) were used as a positive control. After incubation, SytoxGreen dye (Molecular Probes, Eugene, OR) was added to each cell sample at a final concentration of 1 μM. Samples were visualized under a fluorescence microscope (Nikon Eclipse-Ti). NET production was also visualized using antibodies against myeloperoxidase or elastase (Abcam, Cambridge, UK).

The following materials were obtained from Sigma-Aldrich (St. Louis, MO, USA): 5-HT, N-(3-dimethylaminopropyl)-Nʹ-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS), lactoperoxidase (LPO). Fe₃O₄@PEG-COOH was purchased from Nanjing Nanoeast Biotech Co., Ltd. (Nanjing, China). Vivotrax and Perimag were purchased from Magnetic Insight Inc (Alameda, USA) and Micromod Partikeltechnologie GmbH (Rostock, Germany), respectively. Cy7-NHS was obtained from AAT Bioquest (Sunnyvale, CA, USA). RAW 264.7 cells (Otwo Biotech Inc., Shenzhen, China), Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich), fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), and cell counting kit-8 (CCK-8; Beyotime Institute of Biotechnology, Shanghai, China) were obtained for cell experiments. Myeloperoxidase (Abcam, Cambridge, UK), glucose oxidase (Dalian Meilun Biotechnology Co., Ltd., Dalian, China), Matrigel (Corning, Inc., Corning, NY, USA), 4-ABAH (Sigma-Aldrich), and ExiTron (TM) nano 12000 CT contrast agent (Miltenyi Biotec, Bergisch Gladbach, Germany) were purchased for animal experiments. Erythropoietin protein (EPO), rabbit anti-mouse MPO, rabbit anti-mouse CD68, rabbit anti-mouse anti-ɑ-smooth muscle actin (SMA), rabbit anti-mouse CD31, and rabbit anti-mouse MCP-1 antibodies were obtained from Abcam.

Formalin-fixed tissues were embedded in paraffin and sectioned into 5 μm sections. Sections were deparaffinized and rehydrated into PBS. Antigen retrieval was carried out whenever indicated by the antibody manufacturer; native peroxidase activity was squelched using 0.4% hydrogen peroxide. Blocking was carried out using 5% bovine serum albumin in PBS and sections were incubated with the following antibodies at the manufacturer-indicated dilutions: CD31 (Novus Biologicals, Centennial, CO, USA, AF3628, 10 µg/mL), CD206 (Cell Signaling Technology, Danvers, MA, USA, #24595, 1:200), Ki67 (Novus Biologicals, Centennial, CO, USA, NB110-89717, 1:250), Myeloperoxidase (Abcam Waltham, MA, USA, AB 300650, 1:1000), and goat anti-human IgG biotinylated antibody (Vector Laboratories, Newark, CA, USA, BA-3000-1.5, 1:500). Species-specific, HRP-conjugated anti-antibody polymers and DAB+ reagent (both—Cell Signaling) were used to visualize unlabeled primary antibody binding and HRP-streptavidin reagent (SA-5704, Vector Laboratories) was used to visualize anti-human IgG antibody; all sections were counterstained with hematoxylin. Representative images (n = 6 per tissue specimen) were captured using an Olympus BX51 (Center Valley, PA, USA) equipped with Olympus DP72 digital camera and used for statistical analyses. Staining intensity and/or positive staining events were analyzed using ImageJ.

Immunohistochemistry of 8μm skin cryosections was performed using the fluorescence microscope Olympus IX81 (Olympus, Tokyo, Japan) and the TSA Cy3 (PerkinElmer, Waltham, MA) as recommended by the company. The following primary antibodies were used: F4/80 (BD Pharmingen), myeloperoxidase (Abcam, Cambridge, MA). The slides were incubated for 30 minutes at room temperature with the biotinylated secondary antibody (Dianova, Hamburg, Germany; BD). Nuclei were counterstained with Hoechst 33342 (Invitrogen). Tissues were mounted in Vectashield H-1000.

The following antibodies were used for western blotting and immunostaining: YAP (Santa Cruz, catalog no. sc-376830 and Sigma, catalog no. Y4770), pYAP (Ser127) (Cell Signaling, Catalog no. 4911), TAZ (Santa Cruz, catalog no. sc-48805 and Santa Cruz, Catalog no. sc-518026), IL6 (Santa Cruz, catalog no. sc-57315), Nos2 (Santa Cruz, catalog no. sc-7271), HDAC3 (Santa Cruz, catalog no. sc-376957), NCoR1 (Thermo Fisher, catalog no. PA1-844A), Myc (Sigma, Catalog no. C3956), FLAG (Sigma, Catalog no. F1804), IL1β (Santa Cruz, catalog no. sc-7884), Arg1 (Santa Cruz, catalog no. sc-166920), Collagen I (Novus Biologicals, USA,catalog no. NB600-408), Emcn (Santa Cruz, catalog no. sc-65495), CD31 (BD Bioscience, catalog no. 553370), Ki67 (Abcam, catalog no. ab16667), CD68 (AbD Serotec, United Kingdom, catalog no. MCA1957), TGFβ (Cell Signaling, Catalog no. 3711), β-galactosidase (MP Biomedicals, USA, catalog no. 863365), β-actin (Santa Cruz, catalog no. sc-47778), PCNA (Santa Cruz, catalog no. sc-56), WGA (Thermo Fisher, catalog no. W11261), Lectin (Thermo Fisher, catalog no. L21409), and Myeloperoxidase (Abcam, catalog no. ab9535).