Anti cd163 antibody

The primary antibodies used for western blotting and immunofluorescence (anti-elastase antibody, anti-histone H3 antibody, anti-myeloperoxidase antibody, anti-CCR7 antibody, anti-CD163 antibody, anti-CD86 antibody, anti-CD206 antibody), anti-iNOS antibody, anti-Arg-1 antibody, and the antibodies of flow cytometry (mouse anti-human CCR7-FITC, mouse anti-human CD86-FITC, mouse anti-human CD163-PE, and mouse anti-human CD206-PE) in this study were all purchased from Cell Signaling Technology (Massachusetts, USA). Both horseradish peroxidase-labeled goat anti-rabbit and anti-mouse secondary antibodies were obtained from Beyotime (Jiangsu, China). The immunofluorescence secondary antibodies were obtained from Biolegend (San Diego, USA). Other chemicals were purchased from Dingguo Changsheng (Beijing, China). The plasmids used in our study were from Escherichia coli. AFB1 (purity > 98%) was purchased from Sigma (Missouri, USA).

Tumor biopsies were fixed in 3.7% neutral-buffered formaldehyde and embedded in paraffin according to standard protocols. Two-micrometer sections were prepared, and morphology was visualized by standard H&E staining. For immunohistochemistry, the activity of the endogenous peroxidase was blocked with 1% hydrogen peroxide, and after antigen retrieval (citric acid buffer, pH 6) at 100°C, sections were incubated with anti-CD68 (dilution 1:250, clone KP1, ThermoFisher Scientific, Waltham, MA, USA), anti-CD86 (dilution 1:75, clone E2G8P, Cell Signaling Technology, Beverly, CA, USA), and anti-CD163 antibody (dilution 1:250, clone D6U1J, Cell Signaling Technology, Beverly, CA, USA), respectively. This was followed by incubation with EnVision™+Dual Link System-HRP (Dako, Carpinteria, CA, USA). Diaminobenzidine (Cell Signaling Technology, Beverly, CA, USA) was used as a chromogen. Sections were counterstained with 1% Mayer’s hematoxylin. Slides were analyzed using a Leica dm2500 microscope equipped with LAS version 4 software (Leica, Germany).