Immunohistochemical Analysis of Thoracic Aorta

As described previously 19 (link), the thoracic aorta was fixed in 4% paraformaldehyde overnight, and then processed, embedded in paraffin and sectioned at 4 μm. The deparaffinized, rehydrated section from thoracic aorta and cryosections from aortic root (5 μm) were microwaved in citrate buffer for antigen retrieval. Sections were incubated in endogenous peroxidase (DAKO, Via Real, Carpinteria, CA) and protein block buffer, and then with primary antibodies indicated overnight at 4°C. Slides were rinsed with washing buffer and incubated with labelled polymer-horseradish peroxidase secondary antibodies followed by DAB⁺ chromogen detection (DAKO). After final washes, sections were counterstained with haematoxylin. All positive staining was confirmed by ensuring that no staining occurred under the same conditions with the use of non-immune rabbit or mouse control IgG.