F4 80

The sequences, preparation and use of the small interference RNAs (siRNAs) used in this work have been already described [16 (link), 43 (link)]. The commercial synthetic siRNAs (siCD40 sense 5′-GUGUGUUACGUGCAGUGACUU, siCD40 antisense 5′-AAGUCACUGCACGUAACACACUG, siSC sense 5′-ACUACAAGACUCGUGACCAUU, siSC antisense 5′-UGGUCACGAGUCUUGUAGUUU, all from Mycrosynth, Switzerland) included stabilizing phosphorothioate backbone and 2′-O-methyl sugar modifications, as well as a cholesterol molecule added at the 3′ end of the sense strand with a pyrolidine linker to facilitate uptake and internalization.
For the immunochemical work, we used a rat monoclonal antibody against F4/80 (Hycult biotech, Uden, NL), a rabbit polyclonal anti-NF-κB-p65 (anti-phospho-S536, Ab86299, Abcam, Cambridge, UK), and a goat polyclonal anti-CD31/PECAM-1 (platelet-endothelial cell adhesion molecule-1) antibody (M-20, sc-1506, Santa Cruz Biotechnology, Santa Cruz, CA). As a secondary antibody we used a goat anti-rat IgG2 (Novus Biologicals, Littleton, CO), a biotinylated horse anti-goat IgG (BA-9500, Vector, Burlingame, CA) or a Vectastain Elite ABC kit for rabbit antibodies (PK-4001-NB, Vector Laboratory, Burlingame, CA).

In this work we used the following reagents from the stated suppliers: enalapril (E6888, Sigma-Merck, St Louis, MO, USA), Angiotensin II (A9525; Sigma-Merck, St Louis, MO, USA), isoflurane (PDG96236, Baxter Corporation, Deerfield, IL, USA), Hematoxilin (HHS32-1L; Sigma-Merck, St. Louis, MO, USA), Oil-Red-O (O0625; Sigma-Merck, St. Louis, MO, USA) DAB (MKCK2487, Sigma-Merck, St. Louis, MO, USA), CV Mount mounting medium (14046430011, Leica Biosystems, Wetzlar, Germany), Masson trichromacy (1004850001; Sigma-Merck, St Louis, MO, USA), F4/80 (HM1066 Hycult Biotech, Uden, The Netherlands), anti-NF-κB (ab7970 Abcam, Cambridge, UK), Goat polyclonal antibody Vectastain ABC Kit (PK-4000, Vector laboratories, Los Angeles, CA, USA), and IgG2a goat anti-rat (NB7122, Novus Biologicals, Centennial, CO, USA) C57BL/6J ApoE^−/− mice (Jackson Laboratories, Bar Harbor, ME, USA).

Paraffin tissue sections were stained for CD3 (Abcam, Cambridge, UK), CD45R (Invitrogen, Waltham, MA, USA), and F4/80 (Hycult Biotech, Uden, The Netherlands). Sections were dewaxed with xylene and rehydrated with a decreasing battery of ethanol solutions until distilled water. Then, sections were blocked and immunoperoxidase labeled using a EnVision Dual Link Kit (Dako/Agilent, Santa Clara, CA, USA) in accordance with the manufacturer’s protocol. Peroxidase-conjugated secondary antibody staining was followed by diaminobenzidine substrate development, controlling the incubation time under the microscope. To quantify CD3 and CD45R expression, at least 15 high-power fields were counted, and the mean value was expressed. For quantifying F4/80 expression, a semiquantitative intensity score from 0 to 3 was used.