Provis ax 80 microscope

Following the dissection, the aortas were thoroughly flushed several times with ice-cold saline, and subsequently embedded and frozen in Tissue-Tek O.C.T. Compound (Sakura Finetek, Torrance, CA). Frozen aorta tissue specimens were cut at 6 μm-thick-sections on a cryostat, fixed in ice-cold acetone for 20 min and washed for 5 min in phosphate buffered saline (PBS) (pH 7.4). Sections were incubated with blocking buffer (Protein Block Serum-Free, Dako, CA) for 10 min at room temperature, and then incubated overnight with goat anti-adiponectin (dilution, 1:100; R&D systems Inc., Minneapolis, MN) or control goat IgG in PBS containing 1% fetal bovine serum (FBS). Labeling was visualized by biotinylated anti-goat IgG antibody (1:200; Vector Laboratories, Burlingame, CA) and avidin-biotin-horseradish peroxidase procedure (Vectastatin ABC kit, Vector laboratories) with 3,3′-diaminobenzidine tetrahydrochloride (DAB, Sigma-Aldrich, St Louis, MO) according to the protocol recommended by the manufacturer. To confirm atherosclerotic plaque formation, sections from ApoE-KO mice were stained with hematoxylin and eosin (H & E) or Oil Red O. Light microscopy was carried out with a Provis AX-80 microscope (Olympus, Tokyo, Japan).