Ab65871

Formalin-Fixed, Paraffin-Embedded (FFPE) tongue and jejunum samples were sectioned onto Superfrost slides (Thermo Fisher Scientific, MA, USA), followed by deparaffinization and rehydrated according to standard protocols. Slides were then prepared for IHC with anti-Myeloperoxidase (MPO) (ab65871, Abcam, Australia) rabbit polyclonal antibody using mouse and rabbit specific HRP/DAB IHC detection kit—micro-polymer (ab236466, Abcam, Australia) according to manufacturer's instructions. Briefly, rehydrated slides were treated with hydrogen peroxide (Abcam, Australia), which was followed by heat-induced epitope-retrieval (HIER) with 0.1 M citrate buffer for 15 min. The sections were then incubated with the protein-blocking reagent (Abcam, Australia) for 10 min and then treated with a specific antibody for MPO (Abcam, Australia; 1:1250; 4 °C for overnight). Post PBS washing, the sections were incubated with goat anti-rabbit IgG secondary antibody (Abcam, Australia) at room temperature for 15 min and developed using HRP-conjugated DAB substrate (Abcam, UK), followed by counter-staining with hematoxylin. Grading was performed based on positive stain counting on the scanned digital images of the slides using QuPath open-source digital software v. 0.2.01^{125 (link)}.

For immunofluorescence analysis, cells grown on fibronectin-coated glass coverslips or frozen aortic sections were fixed with 4% PFA for 15 min, permeabilized with 0.1% Triton X-100, and blocked with 10% goat serum at room temperature for 1 h. Samples were incubated with anti-rabbit citH3 antibody (for citrulline R2, R8, and R17; 1:200, ab5103, Abcam, USA), anti-rat Ly6G (1:200, ab25377, Abcam, USA), anti-rabbit-NE antibody (1:200, ab21595, Abcam, USA), and anti-rabbit-MPO antibody (1:200, ab65871, Abcam, USA) at 4°C overnight. On the 2nd day, slides were incubated with goat anti-rabbit/rat TRITC/FITC secondary antibody for 1 h at room temperature and mounted with DAPI (4′,6-diamidino-2-phenylindole, Vector, ZsBio, Beijing, China). Images were obtained using a confocal microscope. At least five fields for each coverslip were captured. The image analysis was performed with ImageJ (NIH, Bethesda, MD, USA).

For immunohistochemistry, colonic samples were deparaffinized and rehydrated, and antigen unmasking was performed by boiling (98 °C) in 10 mmol/L citrate buffer for 30 min. Thereafter, sections were incubated for 10 min in 3% (vol/vol) in hydrogen peroxide to inhibit endogenous peroxidase activity and blocked with normal horse serum for 20 min to minimize nonspecific binding of the primary antibody. Sections were washed with phosphate buffered saline (PBS) with 0.05% Tween 20 (Calbiochem, Darmstadt, Germany) and then incubated overnight at 4 °C with the following primary antibodies: anti-macrophage-associated antigen CD163 (mouse anti-rat CD163, clone ED2, BioRAD; MCA342GA, 1/1000) to quantify the number of proinflammatory macrophages and anti-myeloperoxidase (MPO) to quantify neutrophils (rabbit anti-rat myeloperoxidase antibody carboxyterminal end; Abcam; ab65871; 1/1000).
The peroxidase kit ImmPRESS^® HRP Universal (Vector Laboratories Inc., Burkingame, Burlingame, CA, USA; horse anti-rabbit IgG plus polymer kit or horse anti-mouse IgG PLUS polymer kit) was used as secondary antibody. Samples were counterstained with hematoxylin and coverslips mounted with Eukitt mounting media (O. Kindler GmbH & Co., Freiburg, Germany). To determine the level of non-specific staining, negative controls were assessed.