Dylight 549 donkey anti rabbit

Abdominal aortic tissues of mice were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into 5-μm sections. Then the paraffin in the abdominal aorta slices was removed and these slices were rehydrated. After being fixed with 95% ethanol at -20ºC for 30 min, the treated VSMCs in each group were then blocked with PBS containing 5% BSA at room temperature for 60 min. For detecting the target protein expression, samples were incubated with anti-CD68 primary antibody (1:500; ab53444, Abcam Inc., Cambridge, MA, USA) and anti-α-SM-actin primary antibody (1:50; ab179467, Abcam Inc., Cambridge, MA, USA). Additionally, rabbit IgG was also used here to serve as the NC. Nucleus was stained with 4’6-diamidino-2-phenylindole (DAPI) or 3, 3'-diaminobenzidine (DAB). After incubation with the primary antibodies, the slices were washed with PBS for three times and subsequently incubated with DyLight 549-donkey anti-rabbit (SA5-10064, Invitrogen, Carlsbad, California, USA) supplemented with 5% BSA-PBS under dark conditions at room temperature for 2 hours. Finally, the slices were washed, sealed, and observed under the laser confocal microscope.

Upon reaching 40%‐50% confluence, cells in each well were washed 3 times with pre‐cooled PBS (5 minutes each) and fixed with 1 mL of 95% pre‐cooled ethanol at −20°C for 30 minutes. The 95% ethanol was then removed and 1 mL of PBS containing 5% BSA was added for incubation for 60 minutes at room temperature. Then, the cells were incubated overnight at 4°C with 200 μL rabbit polyclonal antibody to CD11b (ab128797, 1 μg/mL, Abcam), rabbit monoclonal antibody to Iba‐1 (ab178847, 1:100, Abcam), rabbit monoclonal antibody to NeuN (ab177487, 1:300, Abcam), rabbit polyclonal antibody to GFAP (ab7260, 1:1000, Abcam) or rabbit polyclonal antibody to CC1 (ab15270, 1:800, Abcam) or rabbit IgG (serving as NC). Thereafter, the cells were incubated with DyLight 549‐donkey anti‐rabbit (SA5‐10064, Invitrogen) at room temperature for 2 hours without light exposure, mounted with glycerine and observed under a laser confocal microscope.