Anti cd206 antibody

BV-2 cells (2 × 10⁴) were cultured on coverslips and then incubated with LPS (50 μg/ml) or Gps for 48 h. Cells were then fixed in 4% paraformaldehyde for 30 min and treated with 0.1% Triton X-100 for 20 min, followed by blocking with 5% BSA (bovine serum albumin) for 30 min at room temperature. Cells were incubated with anti-iNOS antibody (ab283655, Abcam, Cambridge, MA, United States) or anti-CD206 antibody (#24595, Cell Signaling, United States) overnight for 4°C. After three washings with ice-cold PBS, the cells were incubated with a secondary antibody [Cy3 conjugated Goat Anti-Rabbit IgG (H + L), GB21303, Servicebio, China] for 1 h at room temperature. After three washings, the nuclei were counterstained with DAPI (4’,6-diamidino-2-phenylindole) for 10 min at 37°C. Different microglial state markers were observed using a fluorescence microscope [Olympus, Tokyo, Japan, numerical aperture (NA = 1.4)] and photographed at × 400 magnification. The mean fluorescent intensity was measured by ImageJ software.

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).