Mpo antibody

Memantine hydrochloride and the NETosis assay kit were purchased from Cayman Chemical (Ann Arbor, MI). The NET activator PMA, phagocytosis inhibitor Cytochalasin D, MPO antibody, and a S100A9 antibody were purchased from Abcam (Cambridge, MA). The neutrophil elastase (NE) ELISA Kit was purchased from R&D Systems (Minneapolis, MN). DNA fluorescent dye Picogreen was purchased from Thermo Fisher Scientific (Waltham, MA), CHRNA7 (α7R encoding gene), and S100A9 siRNA (small interfering RNA) kits were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The CHRNA7 antibody was purchased from GenScript (Piscataway, NJ). The S100A9 ELISA kit was purchased from CUSABIO (Wuhan, China).

Myeloperoxidase (MPO) is a heme-peroxidase that mostly exists in neutrophils, making up roughly 5% of the total dry cell weight of neutrophils, while other cell types such as monocytes and certain macrophage subpopulations contain a much lesser extent of MPO [32 (link)]. Therefore, in this study, we used MPO to target neutrophils. MPO expression levels in the small intestine and colon were assessed by immunohistochemistry. Paraffin sections were dewaxed by successive immersions in xylene and ethanol. The antigen was then repaired at high temperature and pressure using 1× citric acid (pH 6.0) as the repair solution. The sections were incubated with 10% goat serum for 30 min at 37 °C and with MPO antibody (Abcam, Cambridge, England) overnight at 4 °C. The secondary antibody was prepared in PBS-Tween 20 at a dilution ratio of 1:100 and incubated at 37 °C for 1 h. Finally, DAPI working solution was added dropwise to the tissue and incubated at room temperature for 5 min, then washed with PBS three times for 5 min. The liquid was shaken dry, and an anti-fluorescent blocker was added dropwise to the tissue and covered with a coverslip.

After deparaffinization, rehydration, and antigen retrieval, sections were incubated with myeloperoxidase (MPO) antibody (1 : 50; Abcam, Cambridge, UK) for 1 h at room temperature, followed by incubation with goat anti-rabbit secondary antibody, and then visualized with a diaminobenzidine kit. MPO-positive cells were counted in 5 high-power fields (HPFs)/section under a microscope (×400), and the number of cells/field was shown.

Protein lysates were obtained from a paw tissue previously pulverized in liquid nitrogen using RIPA buffer and subsequently subjected to Western blot analysis as described by Dinic et al. (2017) (link). Briefly, the extracted proteins (20 μg) were separated on 12% SDS–PAGE and transferred to 0.2 mm nitrocellulose membrane (GE Healthcare) using Bio-Rad Mini trans-blot system (Bio-Rad, Hercules, CA, United States). The membranes were incubated for 2 h with anti-myeloperoxidase (MPO) antibody (1:1000; Abcam) and anti-β-actin (1:1000; Thermo Scientific). The membranes were washed and incubated with appropriate HPR-conjugated secondary antibodies (goat anti-rabbit; 1:10000; Thermo Scientific and goat anti-mouse; 1:10000; Amersham Biosciences, Piscataway, NJ, United States) for 1 h at room temperature. Proteins were detected by enhanced chemiluminescence (Immobilon Western, Merck Millipore). The intensity of the bands was quantified using ImageJ software. The results were normalized to β-actin loading control.

Harvested lungs were inflated and perfused with 10% neutral-buffered formalin. Lung tissue for histopathological analysis was processed and sectioned as described above. Sections were de-paraffinized, fixed, blocked, and immuno-stained with an optimal concentration of a polyclonal rabbit anti-rat myeloperoxidase (MPO) antibody (Abcam, Cambridge, MA) followed by an optimal concentration of biotinylated secondary antibody and then a HRP-streptavidin conjugate reagent. Stain was visualized using 3,3-diaminobenzidine hydrochloride (DAB) as a chromogen. Sections were counterstained with Mayer's hematoxylin solution. Digital brightfield microscope images of immunostained slides were captured using a slide scanner and imaging system software for analysis (Image-Pro Plus versus 7.0, Media Cybernetics, Rockville, MD). Data was expressed as the mean number of MPO⁺ leukocytes from 10 random fields (200x total magnification).

Sections (5 μm) were cut from paraffin blocks and mounted on treated slides (Superfrost plus; Fisher Scientific). Slides were air dried overnight, placed in a 60°C oven for 30 min, deparaffinized in xylene, and run through graded alcohol to distilled water. Endogenous peroxidases were quenched with 0.3% H₂O₂ for 5 min followed by two rinses with distilled water. Slides were pretreated with target retrieval solution, citrate pH 6 (Dako Corporation, Carpinteria, CA, USA), rinsed in distilled water, incubated in Power Block (Biogenex Laboratories Inc, Fremont, CA, USA), rinsed in distilled water, placed in 1x PBS for 5 min then incubated with anti-myeloperoxidase (MPO) antibody (1:2000 dilution, Abcam, Cambridge, MA, USA) for 30 min at room temperature. The slides were rinsed twice in 1x PBS then incubated with a secondary peroxidase-labeled polymer conjugated to goat anti-rabbit IgG (Envision +, Dako Corp.) for 30 min, and then finally rinsed again in 1x PBS. Bound antibody was detected with diaminobenzidine (DAB+ substrate kit, Dako Corp.). Hematoxylin was used for counter-stain. MPO-stained slides were then evaluated by scoring for the presence of neutrophils within the alveolar and interstitial spaces, as described previously [6 (link)].