Alexa flour647 conjugated anti cd68

To detect the polarization phenotype of the macrophages in the abdomen, the PMs were analyzed by flow cytometry. PMs were washed with staining buffer (1% BSA in PBS containing 0.01% NaN₃, Thermo Fisher, United States) and incubated with 10% mouse serum for 20 min on ice. Subsequently, the cells were incubated with reagents from the LIVE/DEAD™ Fixable Dead Cell Stain Kit (Thermo Fisher, United States), FITC-conjugated anti-CD163 (Bio-Rad, United States), and PE-conjugated anti-CD86 (BD Biosciences, United States) at the manufacturer’s recommended dilution for 40 min on ice. For intracellular staining, the cells were fixed and permeabilized with fixation buffer from a Fixation/Permeabilization Solution Kit (BD Biosciences, United States) for 1 h at 4 °C in the dark, washed with permeabilization buffer and incubated with Alexa-Flour647-conjugated anti-CD68 (Bio-Rad, United States) antibody in permeabilization buffer for 1 h at 4 °C in the dark. The samples were then washed and resuspended in permeabilization buffer and analyzed with a FACS Canto II system (BD Biosciences). The results were analyzed with BD FACS DIVA software (BD Biosciences, United States).

The distribution of the two phenotypes of macrophages in the pancreas was assayed by immunofluorescence staining. Pancreatic tissues were washed with PBS twice, fixed with 4% paraformaldehyde for 24 h and dehydrated in a 30% sucrose solution. Then, the tissues were embedded in Tissue Freezing Medium and cut into 7-μm thick sections. The slides were washed with PBS and permeabilized with 0.1% Triton X-100. Subsequently, the slides were incubated with goat serum at 37 °C for 30 min. The samples were stained by incubation with Alexa-Flour647-conjugated anti-CD68 (Bio-Rad, United States) and FITC-conjugated anti-CD163 (Bio-Rad, United States) or Alexa-Flour647-conjugated anti-CD68 and PE-conjugated anti-CD86 (BD Biosciences, United States) at the manufacturer’s recommended dilution at 4 °C overnight; then, the samples were stained with DAPI to visualize the nuclei. The distribution of the two phenotypes of macrophages in the pancreas was examined by laser scanning confocal microscopy.