Histology

Starting at the bifurcation, 10 um thick sections were collected in series of 5 glass slides (SuperFrost Plus slides, Menzel-Glaser, Germany) with 4 sections per glass. For wt mice, each series was separated by 100 um. For morphometry, 1 glas from 3–4 subsequent series were stained with Masson’s trichrome (Sigma-Aldrich, HT15-1KT) according to the manufactures instructions. Intimal and medial areas were quantified and the collagen-positive area was determined as the percentage of blue-staining using the image analyses software Visiopharm (Visiopharm, Hørsholm, Denmark).

To stain macrophages, we performed anti-MOMA-2 immunohistochemistry as previously described³² with the following modifications: Sections were incubated with primary MOMA-2 antibody (rat anti-mouse IgG2b, Serotec, Düsseldorf, Germany, MCA519) diluted 1:300 overnight at 4 °C.

To stain smooth muscle cells, a biotinylated primary antibody (Actin, smooth Ab (clone 1A4); MS-113- B1; Neomarkers, Freemont, CA, USA) was applied. Endogenous peroxidase activity was blocked with 0.5% H₂O₂, rinsed in dH₂O and antigen retrieval was obtained with proteinase K treatment for 2 minutes and rinsed in TBS-T, and unspecific binding was blocked with 2% BSA. Sections were incubated with primary antibody (0.2 ug/ml in 1% BSA in TBS) over night at 4 °C. After rinse in TBS-T, antibody binding was detected with the Vectastain, Elite ABC kit (VWR, Denmark, PK6100) and DAB (Dako), and sections were counterstained with Mayer’s haematoxylin. MOMA-2 and α-SMA staining was scored independently by two investigators from 0–3 based on area and intensity.

For CD31 staining, endogenous peroxidase was blocked with 0.5% H₂O₂, antigen retrieval was obtained with proteinase K treatment, and sections were blocked with 2% BSA in TBS-T for 1 hour. Sections were incubated with primary anti-Cd31 antibody (ab28364, Abcam; 0.5 ug/ml) over night at 4 °C, and the EnVision System HRP-labeled Polymer anti-rabbit (Dako, K4002) followed by DAB (Dako) was used for detection.