Antimouse cd206

Fresh liver tumor tissues isolated from C57BL/6 mice were fixed in 10% formalin, dehydrated, embedded in paraffin and sliced. The sections were then deparaffinized in xylenes and rehydrated by a gradient of alcohols, followed by antigen retrieval and blockage of endogenous peroxidase activity. Tissue sections were then incubated with primary antibodies antimouse CD206 (1:500, PA5-46994, Invitrogen) and CD86 (1:200, PA5-114995, Invitrogen) for 2 hours. Chromogen development was automatically accomplished with EnVision Detection system (Agilent Dako) and images were obtained by Nikon microscope.

Macrophage polarization was detected by flow cytometry. Briefly, cells were incubated with the antibody conjugated with fluorescence in 100 μL of flow cytometry staining buffer (S1001, Multi Sciences) and were protected from light and incubated for 30 min at 20°C‐30°C. The data were analyzed with FlowJo‐V10 CL software. Each assay was replicated three times. Antibodies for flow cytometry: anti‐human CD86 (Cat#305419), anti‐human CD163 (Cat#333603), anti‐human CD14 (Cat#301803) were purchased from Biolegend (San Diego, CA, USA); anti‐mouse CD206 (Product#12‐2061‐80) was purchased from Invitrogen; anti‐mouse CD11c (70‐AM011C05‐100) and anti‐mouse F4/80 (AM04800201) were purchased from Multi Sciences.