Ab90810

Gastric paracarcinoma and cancerous tissues from animal models were fixed with 4% paraformaldehyde for 48 h, dehydrated, and embedded in paraffin. The paraffin sections were subjected to antigen retrieval, sealing, incubation with primary antibodies (1:50 dilutions of myeloperoxidase [MPO] [ab90810; Abcam, UK], cit-H3 [ab5103; Abcam], CD66b [ab207718; Abcam], CCDC25 [21209–1-AP; Proteintech, China], and CD31 [ab228968; Abcam]), incubation with secondary antibodies (1:100 dilutions of horseradish peroxidase [HRP]-labeled goat anti-mouse IgG and HRP-labeled goat anti-rabbit IgG), 4′,6-diamidino-2-phenylindole (DAPI) staining (Solarbio Life Science, Beijing, China), coloration, and sealing. The samples were then observed and photographed using a confocal microscope (LSM800; Zeiss, Germany).

Slides were thawed and washed twice with PBS; blocked and permeabilized with PBS 1% bovine serum albumin, 5% goat serum (Biological Industries, Beit Ha’emek, Israel) and 0.05% Triton-X (Sigma-Aldrich) for one hour; and incubated with the following primary antibodies overnight at 4 °C: αSMA (1/400, 19245, Cell Signaling, Danvers, MA, USA), GFAP (1/1000, ab7260, Abcam), MPO (1/100, ab90810, Abcam), RECA (1/100, ab9774, Abcam). Slides were washed three times with PBS and incubated with a fluorescent-labeled secondary antibody (1/700, Alexa-Flour) for 1 h at RT. For IgG staining, brains were incubated overnight with 1/200 Alexa-Flour Cy3 anti-rat IgG antibody. DNA was stained for 10 min with DAPI (1:1000, Sigma-Aldrich). Slides were mounted with Flouromount-G. αSMA and RECA images were used to calculate vessel area. In order to minimize the background signal, slides were incubated with secondary antibodies and without primary antibodies. This ensures that staining is produced from the detection of the antigen by the primary antibody and not by the detection system or the specimen.

Neutrophils were isolated from peripheral blood of 10 healthy controls, 12 diabetic patients, and 12 DFU patients using Neutrophils isolation kit (Solarbio, Beijing, China). Purity of cells was >95% as determined by Wright–Giemsa staining. Neutrophils were resuspended at 2 × 10⁵ cells per well in RPMI 1640 medium (Hyclone, Thermo Fisher, MA, USA) and plated onto poly-l-lysine-coated coverslips (Sigma-Aldrich, MO, USA) in 24-well culture plates (Costa, Cambridge, USA), which were incubated in serum-free medium for 2 h to prime NETosis spontaneously. Cells were then instantly fixed in 4% paraformaldehyde (PFA) for NET quantification as previously described^{21 (link)}. NETs were stained with anti-citrullinated histone H3 (CitrH3, citrulline R2 + R8 + R17, Abcam ab5103, Cambridge, USA) and anti-MPO (Abcam ab90810) antibodies, followed by incubation with secondary Alexa Flour 488 (Abcam ab150077) and Alexa Flour 647-conjugated antibodies (Abcam ab150115). After DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich), images were collected with Olympus BX51 microscope (Olympus, Tokyo, Japan) and Qimaging camera (RoHs, British Columbia, Canada). Percentage of NETs was determined from five non-overlapping fields per well for every sample, and then the images were merged with Image J software (NIH, MD, USA).

Lungs were removed, fixed in 4% paraformaldehyde for 4 h, and immersed in 30% sucrose for 3–4 days at 4°C. Sections, 15 μm thick, were cut from frozen lung tissues on a cryostat. Rabbit anti-cleaved caspase-3 (Asp175, diluted 1:500 in PBS; Cell Signaling Technology) and mouse anti-MPO (ab90810; diluted 1:100 in PBS; Abcam) were used as primary antibodies. The staining was visualized with a confocal laser scanning microscope (Leica DM IRE2, Germany). MPO and cleaved caspase-3 were used to identify, neutrophils and apoptotic cells, respectively. Double-positive cells were considered to represent apoptotic neutrophils.

After sectioning to 4 µm, the joint section was blocked with 10% normal goat serum for 45 minutes. The sections were incubated with an anti‐citrullinated Histone H3 antibody (Rabbit, 1:250, citrulline R2 + R8 + R17, ab5103; Abcam) and anti‐Ly6G antibody (1:300) or anti‐myeloperoxidase (MPO) (mouse, 2D4, 1:50, ab90810; Abcam). Isotype control antibodies were used. After three washes, Alexa 488‐goat anti‐rabbit (A‐11034; Invitrogen) to recognize the anti‐Histone H3 and Cy3‐goat anti‐rat (A10522; Invitrogen) to recognize the Ly6G were both incubated for 1 hour at room temperature. A 4',6‐diamidino‐2‐phenylindole (DAPI) nuclear stain was applied for 10 minutes followed by three washes of phosphate buffer saline (PBS) to remove residual dye. Slides were analyzed and imaged with a confocal microscope (Nikon Eclipse Ti). NETs or neutrophil initiated NETosis were identified according to the colocalization of DAPI, Ly6G, and NETs marker cit‐H3.

The mice were injected intraperitoneally with recombinant Rv0888NS/MS, D438A/MS, or H481N/MS. Control mice were treated with PBS and recombinant pMV262/MS. After 48 h, the mice were killed, and the right lungs were removed. Frozen 6-µm lung tissue sections were fixed in ice-cold acetone for 15 min. The Vector M.O.M. Immunodetection Kit (Vector Laboratories, Burlingame, CA, USA) was used with some modifications. Briefly, after blocking the sections in M.O.M. Mouse Ig Blocking Reagent at RT for 1 h, samples were incubated with primary anti-MPO antibody (mouse, 1:50, Abcam, ab90810) or anti-histone H3 (citrulline R2 + R8 + R17) antibody (rabbit, 1:400, Abcam, ab5103), followed by incubation with a secondary goat anti-rabbit IgG antibody (Alexa Fluor 488) (1:500, Abcam) and a goat anti-mouse IgG antibody (Alexa Fluor 633) (1:200 ThermoFisher Scientific). Slices were mounted in Fluoroshield with DAPI (Sigma-Aldrich) and analyzed with a Leica TCS SP5 confocal laser-scanning microscope.