Anti cd86 antibody

Frozen sections of mice colon tissues were first fixed in acetone and permeabilized, and then blocked with 5% normal goat serum at room temperature for 1 h before incubating with primary antibody followed by secondary antibody. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) working solution. The fluorescent staining of colonic tissue was observed using a fluorescent microscope (Nikon) and images were obtained using Case Viewer software (version 2.3, 3D Histech). The average intensity of immunofluorescence staining was analyzed semi-quantitatively using ImageJ software. The primary antibodies were used as follows: anti-ZO-1 antibody (SantaCruz Biotechnology), anti-occludin antibody (SantaCruz Biotechnology), anti-F4/80 antibody (Cell Signaling Technology), anti-CD86 antibody (Cell Signaling Technology), anti-CD206 antibody (Cell Signaling Technology) and anti-integrin β6 antibody (Cell Signaling Technology).

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).

Paraffin sections of each group were undergone antigen retrieval with sodium citrate buffer and blocked by 3% goat serum (16210064, Gibco, USA) for 1 h. Diluted primary antibodies (anti-CD31 antibody (Abcam, ab28364); anti-Ki-67 antibody (Cell Signaling Technology, 12202T); anti-CD163 antibody (Abcam, ab182422); anti-CD86 antibody (Cell Signaling Technology, 19589S)) with blocking buffer were used to incubate at 4 °C overnight. After washing with PBS, the cell and tissue staining kit (CTS005, Anti-Rabbit HRP-DAB System, R&D Systems) was used to detect the staining. Images were taken by an inverted fluorescence microscope (Axio observer A1, Zeiss, Germany) and analyzed by ImageJ software.