Rabbit anti mpo

To assess NET formation, neutrophils combined in round coverslips (from 24-well plates) from different groups were fixed with 4% paraformaldehyde for 15 min at 4°C. After washing steps, the nuclei were stained with Hoechst 33342 dye (Beyotime) for 8 min at room temperature. Images were obtained with a confocal laser-scanning microscope (Leica TCS SP8; Leica) with 40×/1.3 numerical aperture differential interference contrast and analyzed with the instrument's software. The percentage of NET with all neutrophils visualized was calculated to assess NET formation.
To assess MPO and NE formation, neutrophils combined in round coverslips (from 24-well plates) from control and SCH cows were fixed with 4% paraformaldehyde for 30 min at room temperature and nonspecific binding sites were blocked with 3% Albumin Bovine V (Biosharp), 5% normal goat serum (Boster), and 0.5% Triton (BioFROXX) in PBS (Biosharp) for 30 min at room temperature. Cells were then incubated with rabbit anti-MPO (1:500, Proteintech)-NE (1:500, Biorbyt) at 4°C in a humidified chamber overnight. Cells were rinsed 4 times with PBS and incubated with Cy3 goat anti-rabbit IgG (1:1,000, Beyotime) for 1 h at room temperature. After 4 washing steps, the nuclei were stained with Hoechst 33342 dye (Beyotime). Images were obtained with a confocal laser-scanning microscope (Leica TCS SP8; Leica).

Haematoxylin and eosin (H&E), immunohistochemical and immunofluorescence analyses were performed as described previously. 18 The primary antibodies used were mouse anti-Ki67 (Proteintech), rabbit anti-MPO (Proteintech), rat anti-F4/80 (Proteintech) for immunohistochemistry and anti-Ki67 (Abcam) for immunofluorescence. The cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). For each stained section, 6-10 images from random fields were taken, and at least 3 mice/ group were subjected to each experiment. Image-Pro Plus 6.0 was used for the image analysis of sections.