Anti hla dr

Paraffin‐embedded slices (5 µm) were used for immunohistochemistry and immunofluorescence. First, the sections were incubated with an anti‐FPR2 (1:500; Abcam) primary antibody overnight at room temperature. Second, these sections were incubated with secondary antibodies conjugated to a peroxidase‐labeled dextran polymer (Envision + System‐HRP; Boster) for 1 h at 37°C. Finally, these sections were stained with hematoxylin, dehydrated, and coverslipped. For double‐labeling immunofluorescence staining, sections were incubated at 4°C for 12 h with the following antibodies: anti‐FPR2 (1:500; Abcam), anti‐NF200 (1:500; Millipore), anti‐glial fibrillary acidic protein (GFAP) (1:500; Sigma), and anti‐HLA‐DR (1:500; Abcam). These sections were then incubated with the corresponding Cy3‐ and 488‐conjugated secondary antibodies (both at 1:500; Invitrogen) for 2 h at 37°C. Next, these sections were incubated with 4′,6‐diamidino‐2‐phenylindole (Beyotime) for 3 min at room temperature. Finally, the fluorescent sections were imaged with a Zeiss Axio Vert microscope (Zeiss).

The IHC staining was performed as described previously.^{[15 (link)]} A series of 5 μm thick paraffin sections from human left atrial appendages were blocked with 2% BSA (bovine serum albumin) in PBS (phosphate-buffered saline) for 30 minutes and then stained with antibodies, anti-HLA-DR (Abcam, Cambridge,UK) and anti-CD 163 (Abcam) overnight at 4°C.^{[16 (link)]} The slides were washed with PBS, incubated with biotinylated secondary antibody for 20 minutes, counterstained with hematoxylin, and mounted with glycerol gelatin. The staining was visualized using the VectaStain ABC-AP Kit (Alkaline Phosphatase, Standard, Burlingame) as per the protocols. PBS served as a negative control.

Cells were incubated with anti-HLA-DR (M1, macrophage cell subpopulation marker, Abcam) or anti-CD206 (M2, macrophage cell subpopulation marker, Abcam) for 20 min at 4 °C according to the manufacturer's procedures. After washing twice with PBS, cells were resuspended in fluorescence-activated cell sorting (FACS) buffer. Then, fluorescence activated cell sorting was performed by using a FACSAria II instrument (BD Biosciences, Franklin Lake, NJ, USA), and the data were analyzed using FACSDiva 6.1.1 software (BD).