Ab195191

The 8-μm frozen sections of cerebral cortex and cells were fixed, permeated with 0.3% Triton X-100, and blocked with 1% bovine serum albumin. Then, sections/cells were incubated with primary antibodies to CD16 (1:200, A13980, ABclonal), CD206 (1:100, ab195191, Abcam), and Iba 1 (1:100, A12391, ABclonal) overnight at 4°C, and incubated with corresponding fluorescent secondary antibodies (AS011 and AS007, ABclonal). Finally, images were captured under a confocal laser scanning microscope (FluoView FV10i, Olympus; 40 ×) in 3 randomly selected fields from each section (5 sections were randomly selected from each mouse). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was adopted for cell counting and fluorescence intensity analysis.

Raw264.7 macrophages and BMDMs (1 × 10⁶ cells/mL) were treated with interferon-γ (IFN-γ) at 20 ng/mL for 2 h. Da-g-Xan at 1, 5, or 20 μg/mL was added, and cells without Da-g-Xan were considered as controls. Twenty-four hours later, cells were collected and incubated with CD11b (0.25 μg/test, Invitrogen, 56-0112-82), CD86 (0.125 μg/test, Invitrogen, 12-0862-82) and CD206 (1 μg/mL, abcam, ab195191). Then, cells were measured using a BD FACSCalibur CellSorting System (BD Bioscience, USA) and analyzed with FlowJo software (Tree Star Inc, USA). This experiment was performed in triplicate. Dissected tissues from rat colonic anastomosis were washed with Hanks Balanced Salt Solution (HBSS) for three times and minced into 1-2 mm pieces. The tissue pieces were digested with 2 mL GEXSCOPETM Tissue Dissociation Solution (Singleron, China), and the red blood cells were removed with 2 mL GEXSCOPETM red blood cell lysis buffer (Singleron, China). The resulting cells were stained with CD 206 (1 μg/mL, abcam, ab125028) with secondary donkey anti-rabbit antibodies (1 μg/mL, abcam, ab150075), CD11b (2 μg/mL, BD Biosciences, cat. 554862), CD86 (2.5 μg/mL, BD Biosciences, cat. 561961) and then measured using the CellSorting System.

The sections were treated with rat anti‐F4/80 (Ab6640; 1:50; Abcam, Shanghai, China), mouse anti‐CD86 (NBP2‐44515, 1:50; Novus, Shanghai, China), and rabbit anti‐CD206 (Ab195191, 1:50; Abcam) primary antibodies (15 hours, 4℃) followed by staining with Cy3‐labeled goat anti‐rat IgG (Bs‐0293G‐CY3; 1:100, Bioss, Beijing, China), FITC‐labeled goat anti‐mouse IgG (BA1101, 1:100; Boster Biological Technology Co, Ltd, Wuhan, China), and FITC‐labeled goat anti‐rabbit IgG (BA1105, 1:10; Boster Biological Technology Co, Ltd) secondary antibodies (1 hour, 20℃), respectively. The sections were then treated with 4,6‐diamino‐2‐phenylindole (DAPI, C1002; Beyotime Biotechnology Co Ltd, Wuhan, China) to stain the nuclei (5 minutes, 20℃). The proportion of M1 and M2 macrophages was evaluated using the mean optical density (MOD) as determined by the ImageJ analysis software.