Mouse anti α smooth muscle actin

PFA-fixed tissue sections were rinsed in phosphate-buffered saline (PBS) with 1% Triton-X100 and then incubated in 10% oxalic acid for 1 hour. For antigen activation, 0.1% trypsin in PBS was added to the tissue sections. Endogenous horseradish peroxidase (HRP) in the tissue sections was blocked using 3% aqueous hydrogen peroxide in methanol for 8 minutes. After washing in PBS, the tissue sections were blocked with Blocking One Histo. The sections were incubated with the appropriate primary antibody overnight at 4 °C. The histological results from the aortic wall were assessed after staining using the following antibodies: rabbit anti-matrix metalloproteinase (MMP) 2 (1:100; Thermo Scientific), goat anti-MMP9 (1:100; Santa Cruz Biotechnology, Inc.), rabbit anti-MCP-1 (1:50; Novus Biologicals), mouse anti-monocytes/macrophages (MAC387) (1:50; Bio-Rad Laboratories), mouse anti-α-smooth muscle actin (1:400; Santa Cruz Biotechnology, Inc.), rabbit anti-CD163 (1:100; Bioss Antibodies). On the following day, the sections were rinsed in PBS, and incubated with the appropriate secondary antibody conjugated to HRP. Slides were developed with DAB (Vector Laboratories, Burlingame, CA, USA), dehydrated in ethanol (80%, 90%, and 100%), cleared in xylene, and covered with a lipid-soluble mounting medium and glass cover slips.