Anti 4 hydroxynonenal antibody

To examine intracellular ROS production in vSMCs and paraffin sections of aorta tissue, we used flow cytometry and immunohistochemistry, respectively. vSMCs (1 × 10⁶) were seeded on a 60-mm flask culture plate and then incubated under 5% CO₂ and 37 °C conditions. Twenty-four hours after the TAK-733 and Ang II treatment, the culture medium was removed, and adherent cells were collected by trypsinization. Cell pellets were resuspended in PBS containing 10 µM CM-H₂DCFDA (Invitrogen, Carlsbad, USA) and incubated under 5% CO₂ and 37 °C conditions for 10 min. After centrifugation at 1800 rpm for 3 min, the cell pellets were resuspended in serum-free media and incubated under 5% CO₂, 37 °C for 10 min. Flow cytometry was performed using an Accuri^TM C6 cytometer (BD Biosciences, San Jose, CA, USA), with 20,000 events recorded for each sample. Paraffin sections were stained with anti-4 hydroxynonenal antibody (1:100; Abcam; Cambridge, UK) and visualized using the Universal DAB Detection Kit (Vector Laboratory, Burlingame, CA, USA). Positive staining was evaluated with light microscopy to assess the histological effects.