Ihc tek antibody diluent

For cryosections, tissue was embedded in optimal cutting temperature, frozen on dry ice, and stored at −80°C. Aortic roots were sectioned on a cryostat to generate 10‐ to 15‐μm sections. Cryosections were fixed in acetone for 10 minutes at −20°C, blocked in IHC Tek antibody diluent (IHC World, Ellicott City, MD) for 1 hour at room temperature, and incubated with the indicated antibodies in IHC Tek antibody diluent buffer. Antibodies were phospho‐NFκB‐P65 (1:100; Cell Signaling Technology, Danvers, MA); FN (1:400; Sigma, St. Louis, MO); VCAM‐1 (1:200; BD Biosciences, San Jose, CA); ICAM‐1 (1:200; Biolegend, San Diego, CA); MMP9 (1:200; Abcam, Cambridge, UK); MMP2 (1:300; Millipore, Burlington, MA); CD45 (1:150; BD Pharmingen); CD68 (1:200; Abcam); SMA (1:200; Sigma); F480 (1:200; Serotec, Hercules, CA). Sections were washed 3 times in PBS and incubated with Alexa fluor 598‐conjugated donkey anti‐rabbit or ‐rat secondary antibody (Invitrogen, Carlsbad, CA) for 1 hour at room temperature. Slides were washed with PBS and mounted in Vectashield with DAPI (Vector Laboratories, Burlingame, CA). Images were acquired using a Nikon 4 laser confocal microscope. For histology, sections were stained with hematoxylin and eosin, Oil Red, or picrosirius red.