Elastic stain kit

Dulbecco’s Modified Eagle’s Medium (DMEM) and cell culture reagents were purchased from ThermoFisher Scientific. Mouse macrophage colony stimulating factor (M-CSF) was purchased from eBioscience (Cat. #14–8983–80). LPS was purchased from Sigma-Aldrich (Cat. # L4391). Recombinant mouse TNFα and IL4 were purchased from R&D Systems (Cat. # 410-MT and 404-ML, respectively). Elastic Stain kit was purchased from Sigma-Aldrich (Cat. # HT25A-1KT).

Van Gieson staining was performed at University of California–San Diego Moores Histology Core. Deparaffinized slides were stained using Elastic Stain Kit (Sigma-Aldrich) as its protocol (procedure no. HT25). Elastic fibers and nuclear components are stained blue to black, collagen is red, and muscle/other tissues are yellow.

Twenty-eight days after Ang II infusion, mice were euthanized and perfused briefly with PBS, followed by prolonged whole-body perfusion with 4% paraformaldehyde solution. After dissection from the surrounding tissue, the suprarenal aorta was further fixed in 4% paraformaldehyde overnight, and then embedded in paraffin. Suprarenal abdominal aortic tissue was sectioned into 5 µm-thick serial sections. To visualize elastic lamina, histological sections were stained with the Elastic Stain kit (Sigma-Aldrich) following manufacturer’s instructions. To evaluate macrophage infiltration, sections were immunostained with a mouse F4/80 antibody. In brief, sections were subjected to heat-mediated antigen retrieval, blocked with 5% horse serum for 1.5 hour at room temperature, and then incubated with rat polyclonal anti-mouse F4/80 antibody (AbD Serotec) overnight at 4 °C. Detection of F4/80+ cells was achieved using the biotin/streptavidin-HRP system (Vector Laboratories). Histological sections were examined under a light microscope (Biorevo, Keyence). Four random microscopic fields in each mouse (n=4 in each group) were counted, and macrophage number was expressed as number of F4/80-positive cells per square millimeter.