Alexa 488 anti mouse f4 80

FACS analysis was performed using routine protocols with a FACS Calibur flow cytometer (BD Immunocytometry Systems, Franklin Lakes, NJ, USA). Antibodies for staining included APC anti-mouse CD11b (Biolegend, San Diego, CA, USA), Alexa 488 anti-mouse F4/80 (Biolegend), biotin anti-mouse Ly6G (Biolegend) and avidin-PE (eBioscience), PE anti-mouse MR (Biolegend). Dead cells were excluded by propidium iodide staining.
For intracellular staining, cells were stained with cell surface makers first, and then washed with PBS. After centrifuge, cell pellets were suspended completely with fixation buffer on ice for 30 min, and then permeabilized with a Permeabilization Buffer (eBioscience). After centrifuge again, cell pellets were washed and stained with PE anti-iNOS (eBioscience) followed by FACS analysis.
To determine ROS production, cells were harvested and labeled with a ROS probe, according to the recommended protocol (Beyotime), followed by FACS analysis. Briefly, the culture medium was removed and the cells were incubated in dark with the redox-sensitive fluorescent dye (DCFH-DA, 10 µM) diluted by serum-free medium at 37°C/5% CO₂ incubator for 30 min. The cells were then washed with PBS and the fluorescent intensity of DCF was detected by FACS. The ROS level was analyzed using the mean fluorescent intensity (MFI).

The part of aorta with maximum diameter were harvested on day 14 and embedded in Tissue‐Tek OCT compound (SAKURA 4583, Sakura Finetek, USA, Inc) The samples were frozen at −80°C. Then, 5‐um sections were fixed in pre‐cold 4% formaldehyde for 15‐20 minutes. The samples were blocked in 5% BSA in PBS for 1 hour. Next, Alexa 488 antimouse F4/80 (Biolegend 123119), antimouse NOS2 PE‐eFluor 610 (eBioscience) and Alexa Fluor 647 antimouse CD206 (MMR) (Biolegend 141711) antibodies were used to detect the expression of the M1 or M2 macrophage markers in the aortas. The incubations were performed at room temperature for 1.5 hour in a humidified box and protected from light. DAPI was used to stain the cell nuclei (blue) at a concentration of 1 µM. ProLong™ Gold Antifade Mountant (ThermoFisher, P36930) was applied to protect the fluorescence from fading. Images were taken using a Carl Zeiss Axio Imager AX10 with ZEN 2011 (blue edition). The fluorescence was measured using digital image analysis software (ImageJ v.1.41, National Institutes of Health). The percentage of iNOS⁺ and CD206⁺ cells relative to the F4/80⁺ cells was calculated and analysed in each group.