Immunohistochemical Analysis of Aortic Tissue and Smooth Muscle Cells

Formalin-fixed, paraffin embedded whole aortic tissue samples were sectioned at 1 μm thickness. Aortic tissue sections and paraformaldehyde-fixed primary aortic SMCs were immunolabeled with polyclonal rabbit antibodies against ASMA (1:100, ab5694, Abcam), which was used as a marker of contractile stress fibers, or vimentin (1:100, ab137321, Abcam), a marker of synthetic SMCs (19 (link)). To assess for cellular senescence, aortic sections and SMCs were immunolabeled with monoclonal mouse antibodies against p16INK4a (1:50, MA5–17054, Invitrogen) and p21 (1:50, MA1–33926, Invitrogen), cyclin-dependent kinase inhibitors that delineate two core senescence initiation pathways (20 (link), 21 (link)). Bound primary antibodies were visualized using fluorescent-conjugated secondary antibodies: goat anti-rabbit Alexa–594 (for ASMA and vimentin), or goat anti-mouse Alexa–594 (for p16 and p21). Aortic sections were counterstained with DAPI before mounting. For the SMCs, coverslips were mounted with DAPI-containing mounting media (Vectashield^®, H–1200–10).