Ab189392

Approximately 40 mg brain or liver tissue were lysed in RIPA buffer including protease inhibitors. Then the tissues were homogenized and centrifuged (12,000 rpm, 5 min, 4 ℃) to collect the supernatant. The concentration of protein was measured by bicinchoninic acid (BCA) method. 40 µg protein samples were separated by 12.5% SDS-PAGE and transferred to PVDF membranes. The antibodies used were as below: RORγt 1:2000 (Abcam, ab207082), Foxp3 1:1000 (Abcam, ab215206), IL-17A 1:3000 (Abcam, ab189377), GM-CSF 1:1000 (Proteintech, 17762-1-AP), MIP-3α 1:1000 (Abcam, ab106151), IL-10 1:1000 (Abcam, ab189392), TGF-β1 1:1000 (Abcam, ab179695), IFN-λ2 0.1 µg/mL (R&D, AF4635), CYP27A1 1:1000 (Abcam, ab126785), CYP7B1 1:1000 (ABclonal, A17872), APP 1:1000 (Abcam, ab126732), and SAA 1:1000 (Abcam, ab199030). Image System Fusion FX (Vilber Lourmat, Paris, France) was used to detect the protein density.

Western blot was performed according to the manufacturer's specifications. The primary antibodies used were MVH (ab27591, Abcam, USA), OCT4 (ab18976, Abcam, USA), TNF-α (EPR22598-212, Proteintech, USA), IL-2 (26,156–1-AP, Proteintech, USA), IL-10 (Abcam, ab189392), TGF-β(ab179695, Abcam, USA). SIRT1(ab12193, Abcam, USA), SIRT3 (ab189860, Abcam, USA), p21(28,248–1-AP, Proteintech, USA), p53 (10,442–1-AP, Proteintech, USA), GAPDH (ab181602, Abcam, USA). All the HRP secondary antibodies were obtained from Affinity Biosciences. After accordingly antibody incubation and washing with TBST, PVDF membranes were imaged using an EasySee Western blot kit (DW101–01, TransGen, China). Images AI600 and Image J were used to scan and analyze the images.

Tissues from the amputation wound and dorsal cutaneous and tail wounds were collected on days 7, 14, and two months after the surgery and aerosol GCB treatment. Tissue sections were formalin fixed and paraffin embedded. Decalcification was performed for the amputation wound.
Mast cell numbers were quantified using toluidine blue staining and counting the number of toluidine blue positive cells in 10 high-power field (h.p.f) under a 20x objective lens.
The total number of macrophages was quantified using anti-F4/80 antibody staining (ab6640, Abcam USA). M1 macrophages were identified and quantified using iNOS staining (PA5-16855, Thermo Fisher Scientific). Anti-mannose receptor antibody (ab 64,693, Abcam USA) was used to identify and quantify M2 macrophages. CD31 antibody (ab 28,364, Abcam USA) was used for endothelial cell staining; anti-VEGFR-2 (ab39256, Abcam USA) was used for VEGF staining; IL-10 and FGF-2 were measured using ab189392 (Abcam USA) and sc-1360 (Santa Cruz Biotechnology), respectively. Ki67 antibody (ab15580, Abcam USA) was used for cell proliferation and fibroblast activation; protein alpha antibody (ab53066, Abcam USA) was used for fibroblast staining. All the sections were incubated with the primary antibody overnight at 4°C in accordance with the protocols provided by companies.

Samples were lysed with RIPA buffer and centrifuged (10 min, 1,049x g) to remove cell debris. Protein concentrations were determined by BCA assay (Beyotime Biotechnology, China). Equal amount of proteins (20 µg) was subsequently separated by 12% SDS-PAGE and transferred to PVDF membrane, which was blocked with 5% skimmed milk and incubated with primary antibodies overnight. After washing with PBS with Tween 20 (1 hr, room temperature) and incubation overnight at 4°C with the appropriate primary antibody, membranes were incubated with secondary antibodies for 2 hr. The following antibodies were used: T-bet (1 : 1,000, ab91109, Abcam), GATA-3 (1 : 1,000, ab214804, Abcam), IL-2 (1 : 200, ab231441, Abcam), IL-4 (1 : 1,000, ab11524, Abcam), IFN-γ (1 : 4,000, AMC4739, Invitrogen), IL-10 (1 : 1,000, ab189392, Abcam), second antibody (1 : 2,000, CST 7074), and β-actin (1 : 2,000, TA-09, Zhongshan Company). Protein bands were visualized using a ChemiDoc Touch imager (Bio-Rad, USA). The gray area ratios of different bands were calculated by densitometry using ImageJ v1.8.0 software (National Institutes of Health). Each experiment was performed in duplicate.

Four-micron thick paraffin sections of kidneys were deparaffinised, rehydrated and rinsed. Heat-mediated antigen retrieval method with sodium citrate buffer (10 mM sodium citrate, 0.05% tween 20, pH 6.0) was performed. Sections were then quenched with 3% H₂0₂ for 20 minutes and incubated with 10% (rabbit/goat) serum for 30 minutes to block non-specific binding. Sections were incubated with rat monoclonal to IL-10 (ab189392; Abcam, Cambridge, UK) or rabbit polyclonal to PAI-1 (ab66705; Abcam, Cambridge, UK) antibody (1:100) overnight at 4 °C. The next day, sections were washed and incubated with anti-rat/anti-rabbit secondary antibody (1:200) for 30 minutes at room temperature. This was followed by incubation with horseradish peroxidase-conjugated streptavidin (VECTASTAIN Elite ABC staining kit; Vector Laboratories). 3,3′-diaminobenzidine tetrahydrochloride was then used to visualise peroxidase conjugates in samples. All slides were then hydrated, cleared and mounted with DPX. Ten random fields per animal were imaged in the renal cortical region using the Olympus BX43 microscope (x20 magnification). Staining was quantified by counting the number of positively stained cells per field. All assessments were performed in a blinded manner.