Anti cd206

We used the RIPA Lysis Buffer (Fudebio, Hangzhou, China) containing phosphatase/protease inhibitors (Fudebio, Hangzhou, China) to extract total protein from the cultured cells following the manufacturer’s method. SDS‐polyacrylamide gel electrophoresis (10%; SDS‐PAGE) was performed to separate the protein samples (20 µg), which were then transferred to Millipore polyvinylidene difluoride (PVDF) membranes. These PVDF membranes were kept in overnight incubation at 4°C with anti‐CD206, anti‐ODC, anti‐IL‐33, anti‐iNOS, anti‐CD68, anti‐ARG1 and anti‐GADPH primary antibodies (Affinity, USA), followed by incubation at room temperature for 60 minutes with horseradish peroxidase (HRP)–conjugated secondary antibodies (Affinity, USA) that were diluted to 1:5000 in 5% skim milk. After washing with TBST, the membranes were treated for 1 minutes with SuperSignal™ West Dura Extended Duration Substrate (Fudebio, Hangzhou, China) and were visualized through chemiluminescence.

On day 7 after surgery, the intestinal anastomotic tissues were collected and fixed with 4% paraformaldehyde (Servicebio, China) after PBS rinses. Next, the anastomotic tissues were embedded with paraffin and sliced into 5-μM-thick sections (surgical anastomotic tissues after repair) for later use. Histological sections were subjected to hematoxylin and eosin (HE) staining and Masson-Trichrome-Goldner (MTG) staining as per standard protocols to assess the level of inflammation, granulation tissue formation, and collagen expression in the anastomotic tissues.
Cell proliferation, angiopoiesis, and macrophage polarization in tissues were assessed by immunofluorescence. Sections were probed using Anti-CD86 (1:200; Affinity, Jiangsu, China), anti-CD206 (1:200; Affinity), anti-CD31 (1:500; Servicebio, Wuhan, China), anti-α-SMA (1:1,000; Servicebio), and anti-Ki-67 (1:1,000; Servicebio), followed by incubation with corresponding secondary antibodies at room temperature. Ultimately, the tissues were counterstained with DAPI. Images were recorded with a fluorescence microscope (OLYMPUS, 1X71 + DP71).