Rabbit anti mpo monoclonal antibody

Lung sections were rehydrated and subjected to heat‐mediated antigen retrieval treatment, followed by treated with 3% H₂O₂ for 15 min, then blocked in 3% bovine serum albumin (BSA) for 30 min. Sections were incubated with rabbit anti‐MPO monoclonal antibody (dilution 1:1000, Abcam, Cambridge, USA) or rat anti‐F4/80 monoclonal antibody (dilution 1:200, Abcam) overnight at 4 °C. The following day, the sections were incubated with secondary antibodies conjugated with horseradish peroxidase (1:200, Beyotime) at room temperature for 1 h after repeated washing with PBS. After three times washing, the sections were then incubated with diaminobenzidine (Beyotime) within 5 min and counterstained with hematoxylin. The tissue sections were imaged using the Leica Microscope system.

For histopathologic analysis, 4% paraformaldehyde fixed colon sample was embedded in paraffin, sectioned (4 μm), and stained with hematoxylin and eosin (H&E). Histological scoring was performed in a blinded fashion by an investigator. Colitis scoring was made as a combined score for severity of inflammation (0 = normal; 1 = slight; 2 = moderate; and 3 = severe), depth of injury (0 = normal; 1 = mucosa; 2 = mucosa and submucosa; and 3 = transmural), crypt (damage: 0 = normal; 1 = basal one-third damaged; 2 = basal two-thirds damaged; and 3 = only surface epithelium intact), and percent of involved area (0 = normal; 1 = 1–25%; 2 = 26–50%; 3 = 51–75%; and 4 = 76–100%). For immunohistochemistry, the expression of myeloperoxidase (MPO) and ZO-1 was detected using rabbit anti-MPO monoclonal antibody (Abcam) and rabbit anti-ZO-1 monoclonal antibody (Invitrogen), respectively. Integrated optical density (IOD) values were measured by Image-Pro Plus 6.0 image processing software (Media Cybernetics). Five fields of vision (at magnification of ×400) per section (two sections per specimen) were used for DSS colitis histological scoring and MPO expression quantification.