Dab chromogen detection

The carotid was fixed in 4% paraformaldehyde overnight, and then processed, embedded in paraffin, and sectioned at 4 μm. The deparaffinized, rehydrated were microwaved in citrate buffer for antigen retrieval. Sections were incubated in endogenous peroxidase (DAKO) 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-antimouse/antirabbit antibodies followed by DAB + chromogen detection (DAKO). After final washes, sections were counterstained with hematoxylin. 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.

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.

The thoracic aorta was fixed in 4% paraformaldehyde overnight, embedded in paraffin and sectioned at 4 μm. The deparaffinized, rehydrated sections from thoracic aortas and cryosections from aortic roots (5 μm) were microwaved in citrate buffer for antigen retrieval, incubated in endogenous peroxidase (DAKO) and protein block buffer and then primary antibodies overnight at 4°C. Slides were rinsed with washing buffer and incubated with labelled polymer‐horseradish peroxidase‐conjugated anti‐mouse/anti‐rabbit antibodies followed by DAB+ chromogen detection (DAKO). Data were analysed using Image‐Pro Plus.