F4 80

F4/80 (#SAB5500103) was obtained from Sigma-Aldrich (St. Louis, MO, USA). The TLR4 inhibitor TAK-242 (#614316) was obtained from Calbiochem (San Diego, CA, USA). The PI3K inhibitor wortmannin (#HY-10197) was obtained from MedChemExpress (New Jersey, USA). Collagen II (#28459-1-AP) was obtained from Proteintech Group (Wuhan, China). TLR4 (#AF7017), CD68 (#DF7518), PI3K (#DF6069) antibodies were obtained from Affinity Biosciences (Cincinnati, OH, USA). SP-D (#ab220422), MD-2 (#ab24182), TNF-α (#ab66579), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#ab181602), MMP-13 (#ab84594), and NLRP3 (ab263899) antibodies were obtained from Abcam (Cambridge, UK). Antibodies for NF-κB p65 (#8242), phospho-p65 (p-p65) (#3033), p-Akt (#4060) and Akt (#9272) were procured from Cell Signaling Technology (Boston, MA, USA). Recombinant human SP-D (rhSP-D) (#CSB-YP021175HU) was purchased from Huamei Biotech Co., Ltd (Wuhan, China). ELISA Kits of rat IL-1β (#ELK1272) and TNF-α (#ELK1396) were purchased from ELK Biotechnology (Wuhan, China). SNP (#1008) was purchased from Youcare Pharmaceutical Group Co., Ltd (Beijing, China). All of the other chemicals and reagents were of analytical grade.

To measure the CD31 and F4/80 protein levels, mice were sacrificed and wound tissues were harvested 7 days after treatment. Briefly, the harvested wound tissues were homogenized using Multi-beads shocker^® and added to the T-PER Reagent (Thermo Fisher Scientific) consisting of proteinase and dephosphorylation inhibitors (Thermo Fisher Scientific). Total protein lysates were obtained after the elimination of debris by filtering with 0.22 μm filters (Millipore). Total protein (10 μg) was separated using a mini-protean TGX Stain-Free^TM gel (BioRad), and then transferred to PVDF membranes using the Trans-Blot^® Turbo^TM transfer system (BioRad). After blocking with the PVDF Blocking Reagent Can Get Signal^® (TOYOBO) for 1 hour at room temperature, the membranes were incubated with primary antibodies against CD31, F4/80, or tubulin (Sigma-Aldrich) at 4 °C overnight, followed by the appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 hour. Expression was visualized by chemiluminescence with a digital luminescent image analyzer (LAS-4000, GE Healthcare).

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.

For immunocytochemistry, OECs and microglia were washed with PBS, and fixed in 4% PFA for 20 min at room temperature. After blocked with 3% BSA for 15 min, cells were stained with primary antibodies: P75NTR (ab52987, 1:500; Abcam), iNOS (ab49999, 1:200; Abcam), F4/80 (SAB5500103, 1:100; Sigma), p‐c‐Jun (3270s, 1:5000; Cell Signaling Technology), and p‐NF‐κB (3033t, 1:2000; Cell Signaling Technology) at 4°C overnight. The cells were then stained with corresponding secondary antibodies. A confocal microscope (LSM 800; Zeiss) was utilized for examination and photography.
For immunohistochemistry, slides of injured spinal segments were blocked in 0.01 M PBS containing 3% BSA and 0.1% Triton X‐100 for 20 min. Subsequently, slides were incubated with primary antibodies: Iba1 (019‐19741, 1:1000; Wako), iNOS (ab49999, 1:200; Abcam), Arginase1 (sc‐166920, 1:200; Santa Cruz Biotechnology), GFAP (1:2000; Sigma), NeuN (#24307, 1:200; Cell Signaling Technology) at RT overnight. The slides were then stained with corresponding secondary antibodies, and counterstained with Hoechst 33342 to display nuclei. Slides were photographed by a confocal microscope (LSM 800; Zeiss).

Assays were performed with power SYBR green PCR master mix (Applied Biosystems, Foster City, CA, USA) as previously described^{50 (link)} using the ABI Prism 7300 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Primers for quantification of HIF-2α, CRP, Hepcidin, IL-6, EPOR, TFR, c-MYC, IL-1β, FPN-1, IRAK-4, MYD-88, TGF-β, CD-163, CD-11c, F4/80, Ferritin, TNF-α, IL-10 and β-actin (Sigma-Aldrich, Rehovot, Israel) are summarized in Supplemental Table 2.

Fluorescent immunohistochemistry was performed on cryostat sections using the method as described previously [23 (link)]. Primary mouse monoclonal, rat monoclonal or rabbit polyclonal antibodies raised against calbindin (Swant, Marly, Switzerland; 1:1000), CD68 (Serotec, Raleigh, NC; 1:500), glial fibrillary acidic protein (GFAP; DAKO, Carpinteria, CA; 1:300), glutamine synthetase (Abcam, Cambridge, MA; 1:1000), alpha subunit of G protein (Goα; Millipore, Billerica, MA; 1:1000), hexaribonucleotide binding protein-3 (NeuN; Millipore; 1:100), Neurofilament (SMI32; Covance, Munich, Germany; 1:1000), Npc1 (Abcam; 1:300), alpha isoform of protein kinase C (PKCα; Sigma; 1:1000), tyrosine hydroxylase (Millipore; 1:1000), and a microglia marker (F4/80; 1:200) were used. All primary antibodies are specific to determine their target proteins, which were used as specific markers to identify different types or structures of cells. Alexa 488-labeled (Molecular Probes, Eugene, USA) or Cy3-labeled (Dianova, Hamburg, Germany) secondary antibodies against mouse, rat or rabbit IgG were used. Fluorescence in sections was detected by a confocal microscope (Zeiss LSM780; Jena, Germany). Digital images were adjusted in contrast and brightness with the Photoshop software (Adobe Systems, San Jose, USA).