Nox2 primary antibody

Proteins were separated by polyacrylamide gel electrophoresis in the presence of SDS. After electrophoresis in Tris-glycine buffer, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane. Next, the membrane was blocked in 5% skimmed milk in Tris-buffered saline containing 0.05% Tween-20 (TBST) for 1 h at room temperature. After washing with TBST, the membranes were incubated with primary antibody at 4°C overnight. After washing with TBST, the membrane was incubated with horseradish peroxidase- (HRP-) bound secondary antibody for 1 h at room temperature, then washed with TBST, and finally visualized using a chemiluminescence kit (Beyotime Biotechnology, China). The HDAC4 primary antibody was purchased from ABclonal Technology (China); the p47^phox primary antibody, NOX2 primary antibody, and HRP-bound secondary antibody were purchased from Proteintech (China).

The fresh lung tissues were fixed in 4% paraformaldehyde (PFA). Then, the samples were gradually dehydrated and embedded in paraffin. Organ sections were cut into 5μm in thickness and then deparaffinized and incubated in citrate buffer at 95 °C for 40 min for antigen retrieval and then incubated overnight at 4 °C with NOX₂ primary antibody (1:100 dilution, proteintech, 19013-1-AP). After three washes, tissue slices were incubated with biotinylated anti-mouse IgG (1:200 dilution, Vector Laboratories, CA, USA) for 1 hour at RT and then washed three times, after which streptavidin-horseradish peroxidase conjugates (Vector Laboratories, CA, USA) were added and the slices incubated for 45 min. After three washes with PBS, DAB solution (Vector Laboratories, CA, USA) was added and the slides were counterstained with hematoxylin.