Goat anti rat igg secondary antibody

The coronary arteries and thoracic aorta samples were primarily homogenized on ice using RIPA buffer (Sigma-Aldrich; Merck KGaA). After being completely lysed, the sample homogenate was centrifuged at 12,000 x g for 15 min at 4˚C. The supernatant was preserved for protein quantification using a BCA kit and 15 µg protein/lane was separated by polyacrylamide gel electrophoresis. The separated proteins were subsequently transferred onto polyvinylidene difluoride membranes at 25 V for 30 min and blocked in 5% non-fat milk for 50 min at room temperature. The membranes were incubated with primary antibodies overnight at 4˚C. The primary antibodies obtained from Abcam were as follows: Anti-HOXA9 (cat. no. ab191178; 1:1,000), anti-PF4 (cat. no. ab129090; 1:1,000), anti-E-selectin (cat. no. ab18981; 1:1,000), anti-VCAM-1 (cat. no. ab134047; 1:1,000) and anti-β-actin (cat. no. ab8226; 1:1,000). After washing with TBS-Tween (0.1%) in triplicate, the membranes were incubated with goat anti-rat IgG secondary antibody (Abcam; cat. no. ab150165; 1:5,000) for 1 h at 37˚C. Protein bands were detected using enhanced chemiluminescent reagent (Santa Cruz Biotechnology, Inc.). The density of blots for proteins were normalized to β-actin using ImageJ 2.0 software (National Institute of Health).