Anti mac3 clone m3 84

To detect the presence of inflammatory cells, vessel wall characteristics and effects of garcinol therapy, IHC was performed on paraffin-embedded sections of cuffs harvested after 3 days (ApoE*3-Leiden) or 21 days respectively (PCAF KO and WT mice). Weigert’s elastin staining was used to visualize elastic laminae. Inflammatory cell presence in the vascular wall was visualized using antibodies against leukocytes (anti-CD45 clone 30-F11, BD Pharmingen) and macrophages (anti-Mac3 clone M3/84, BD Pharmingen). Vascular SMCs were stained using anti-smooth muscle actin (SMA, clone 1A4, Dako) antibodies, PCAF was stained using anti-PCAF (ab12188, Abcam) antibodies and CCL2 was stained using anti-CCL2 (clone M-18, Santa Cruz) antibodies.

The phenotypes and the percentages of Mφ were determined by FACS analysis with fluorescence dye‐conjugated antibodies as described previously.²⁵ The cell suspensions (10⁵ cells/sample in PBS with 1% FBS) were incubated in the dark for 30 min at 25°C with the following antibodies: rat monoclonal anti‐F4/80 (clone BM8, Thermo Fisher Scientific, Waltham, MA, USA) for the primary gating of Mφ; rat monoclonal anti‐CD80 (clone 16‐10A1, Thermo Fisher Scientific) for F4/80⁺CD80⁺ M1 Mφ or anti‐CD86 (clone GL1, Thermo Fisher Scientific) for F4/80⁺CD86⁺ M1 Mφ and anti‐CD206 (clone MR6F3, Thermo Fisher Scientific) for F4/80⁺CD206⁺ M2 Mφ or anti‐CD163 (clone TNKUPJ, Thermo Fisher Scientific) for F4/80⁺CD163⁺ M2 Mφ.
The percentages of Mφ were determined through positive staining with anti‐F4/80, anti‐MAC3 (clone M3‐84, BD Biosciences) or anti‐CD11b (clone M1‐70‐15, BD Biosciences). Briefly, after incubation, the cells were washed with PBS, and the positively stained cells were identified using a FACSCanto II cell analyzer (BD Biosciences) and quantified using the FlowJo software (Tree Star Inc., Ashland, OR, USA).