Anti kis antibody

Protein expression in VSMCs and ECs was determined by western blot analysis. The antibodies used were as the following: Anti-KIS antibody (Cat#SAB1300125), was purchased from Sigma Aldrich (St. Louis, MO). Anti-MARCKS antibody (Cat#5607S) and anti-GAPDH antibody (Cat#2118L) were from Cell Signaling Tech. (Danvers, MA). Protein expression was quantitated through densitometry using the Image Station 4000 MMPro (Carestream Health, Rochester, NY). Signal intensity of the protein of interest was normalized to GAPDH signal intensity.

At the predetermined time of euthanasia, the anesthetized mice were perfused through the left ventricle with 4% paraformaldehyde via a sternotomy. The femoral arteries were excised and snap-frozen in OCT compound (Sakura, Torrance, CA). The arteries were sectioned at 5 μm intervals beginning 2.5 mm proximal to the suture. For assessment of intimal hyperplasia formation, the sections were stained with hematoxylin and eosin, then counterstained with Verhoeff-Van Gieson (VVG) stain (Sigma Aldrich, Cat#HT25A; St. Louis, MO) to allow visualization of the internal elastic lamina (19 (link)). To assess MARCKS expression and KIS expression in the femoral arteries, sections were immunostained with anti-MARCKS antibody (EMD Millipore, Cat# AB9298; Billerica, MA), anti-KIS antibody (Sigma Aldrich, Cat#SAB1300125; St. Louis, MO). These sections were counterstained with α-smooth muscle actin (Sigma Aldrich, Cat#2547; St. Louis, MO) and 4’,6-diamidino-2-phenylindole (DAPI) (Sigma Aldrich, St. Louis, MO). Confocal microscopic images of the stained sections were acquired using a Zeiss 510 laser confocal microscope (Carl Zeiss, Germany). Zeiss Zen imaging software was used to collect and analyze the image data.