Mpo monoclonal antibody

The mice were anesthetized with an overdose of pentobarbital (40 mg/kg), and the heart was perfused with 0.1 mol/L PBS (pH = 7.4) containing 4% paraformaldehyde. The brain was cut into 5-μm-thick coronal sections for immunohistochemical staining. In brief, the sections were incubated with the primary antibodies overnight at 4°C, including rabbit anti-myeloperoxidase (MPO) monoclonal antibody (neutrophils marker, 1:800, Abcam, Cambridge, MA, United States), and rabbit anti-Iba1 polyclonal antibody (microglia marker, 1:200, Wako, Japan). The sections were washed with PBS (pH = 7.4) 3 times, 3 min each time. Sections were then incubated with horseradish peroxidase (HRP) combined with the secondary antibody rabbit immunoglobulin G (IgG) antibody (Servicebio, Wuhan, China) for 1 h at room temperature.

The preceding few steps were identical to those used for tissue immunofluorescence (IF). But before blocking, brain sections (n = 6 per group) were incubated for 15 min at room temperature into a mixed solution (3% H₂O₂). After incubation with rabbit anti-myeloperoxidase (MPO) monoclonal antibody (1:500, Abcam, Cambridge, MA, United States) at 4°C overnight, the sections were washed three times in PBS before adding the horseradish peroxidase (HRP)-combined secondary anti-rabbit immunoglobulin G (IgG) antibody (1:800, Servicebio, Wuhan, China) and incubating for 1 h at room temperature on an orbital shaker. Then all sections were exposed to diaminobenzidine (DAB) reagent for 30 min, followed by hematoxylin. After dehydration, the sections were protected with coverslips.

Lung tissues were fixed in 10% formaldehyde solution, embedded in paraffin, cut into 5 μm sections, and then dewaxed, hydrated, and retrieved in citrate buffer. Then, endogenous peroxidase activity and nonspecific binding were blocked. Subsequently, sections were incubated overnight at 4 °C in a humid chamber with myeloperoxidase (MPO) monoclonal antibody (1:1000, abcam). On the second day, the sections were washed in PBS and incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (1:2000, Hangzhou Baoke Biotechnology. Co., Ltd., China) at 37 °C for 30 min. After washing with PBS, the sections were dyed in DAB (3, 3′-diaminobenzidine), redyed in hematoxylin, differentiated in 1% hydrochloric-alcohol solution, dehydrated, and sealed. Presence of buffy or brown diaminobenzidine precipitates is indicative of positive reactivity, which was observed with an optical microscope.