Fizz1

Alveolar macrophages were washed with PBS and lysed with 1X RIPA buffer containing protease inhibitor cocktail (Thermo Scientific). Fifty micrograms of protein lysates were resolved by SDS-PAGE. Membranes were incubated with antibodies against FIZZ1 (Abcam), Arg (BD Biosciences) and CD206 (Abcam) antibodies. Signals were developed with Pierce ECL Western blot detection kit (Thermo Scientific, Rockford, IL).

The mouse pancreatic tissues were harvested 1, 2, or 4 weeks after MSC infusion. First, the mice anaesthetized with an intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg) were perfused with PBS through the left ventricle, followed by 4% paraformaldehyde. Then, the pancreases were isolated, dehydrated with 30% sucrose/PB overnight, and embedded in optimal cutting temperature compound (OCT). Pancreatic sections (6 mm) were sliced by a microtome (Thermo Fisher Scientific) and incubated in a humidified chamber at 4°C overnight with primary antibodies against insulin (1/200, guinea pig, Sigma-Aldrich), glucagon (1/2,000, mouse, Abcam), Pdx1 (1/200, rabbit, CST), CD11c (1/200, mouse, Abcam), IL1β (1/100, rabbit, Abcam), F4/80 (1/200, rabbit, Sigma-Aldrich), and Fizz1 (1/200, rabbit, Abcam). After the sections were washed with PBS, they were incubated for 2 h with a secondary antibody (1 : 500; Alexa Fluor 488/594-conjugated secondary antibodies, Invitrogen) at room temperature. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole, Sigma-Aldrich). The images were captured with a confocal laser scanning microscope (Olympus, Tokyo, Japan). Peritoneal macrophages spread on glass coverslips were fixed with 4% paraformaldehyde. The remaining steps were performed as described above.