Rabbit anti cd163

PFA-fixed tissue sections were rinsed in phosphate-buffered saline (PBS) with 1% Triton-X100 and then incubated in 10% oxalic acid for 1 hour. For antigen activation, 0.1% trypsin in PBS was added to the tissue sections. Endogenous horseradish peroxidase (HRP) in the tissue sections was blocked using 3% aqueous hydrogen peroxide in methanol for 8 minutes. After washing in PBS, the tissue sections were blocked with Blocking One Histo. The sections were incubated with the appropriate primary antibody overnight at 4 °C. The histological results from the aortic wall were assessed after staining using the following antibodies: rabbit anti-matrix metalloproteinase (MMP) 2 (1:100; Thermo Scientific), goat anti-MMP9 (1:100; Santa Cruz Biotechnology, Inc.), rabbit anti-MCP-1 (1:50; Novus Biologicals), mouse anti-monocytes/macrophages (MAC387) (1:50; Bio-Rad Laboratories), mouse anti-α-smooth muscle actin (1:400; Santa Cruz Biotechnology, Inc.), rabbit anti-CD163 (1:100; Bioss Antibodies). On the following day, the sections were rinsed in PBS, and incubated with the appropriate secondary antibody conjugated to HRP. Slides were developed with DAB (Vector Laboratories, Burlingame, CA, USA), dehydrated in ethanol (80%, 90%, and 100%), cleared in xylene, and covered with a lipid-soluble mounting medium and glass cover slips.

The sections were routinely dewaxed to water, incubated with 3% hydrogen peroxide at room temperature for 10 min, placed in citrate buffer at 95°C for 15 min, and cooled to room temperature. The sections were blocked in goat serum for 30 min and incubated overnight at 4°C with the following primary antibodies: rabbit anti‐CX3CL1 (1:500; Bioss), rabbit anti‐CX3CR1 (1:500; Bioss), rabbit anti‐iNOS (1:400; Bioss), rabbit anti‐CD163 (1:500; Bioss), and rabbit anti‐CD11b (1:4000; Abcam). After being washed in PBS solution, the sections were incubated with the secondary antibodies for 1 h. The following secondary antibodies were used: goat anti‐rabbit IgG horseradish peroxidase (HRP) conjugate (ZSGB‐Bio). After being washed in PBS solution, develop color at room temperature with 3,3'‐diaminobenzidine. The nuclei were stained with hematoxylin. The positive area was counted by an unbiased, blinded investigator using ImageJ software.