Immunohistochemical Analysis of Aortic Tissue

Blocks of paraffin-embedded aorta tissue were sectioned to a thickness of 7 µm, placed on a coating slide and dried at 40 °C for 24 h. Slides were then deparaffinized and incubated in 0.3% H₂O₂ (Sigma-aldrich, Missouri, MO, USA) for 30 min. Slides were subsequently rinsed three times with PBS and incubated in normal animal serum to block non-specific binding and incubated with primary antibodies (Table S2) at 4 °C, followed by three additional rinses with PBS. Slides were then treated with biotinylated secondary antibodies from the ABC kit (dilution rate 1:200; Vector Laboratories, Burlingame, CA, USA), incubated for 1 h with blocking solution, and rinsed three times with PBS. Slides were left to react with DAB substrate for 15 min, followed by mounting with a cover slip and dibutylphthalate polystyrene xylene (Sigma-aldrich, Missourti, MO, USA) mounting solution. Images were detected using a light microscope (Olympus, Tokyo, Japan) and quantification of the intensity of the brown color was performed using Image J software (NIH, Maryland, MD, USA) [45 (link),46 (link)].