Rabbit anti il 10

To detect macrophages that had incorporated exosomes, RAW264.7 cells were co-cultured with PKH67- (Sigma-Aldrich; Merck-KGaA) labeled exosomes for 12 h at 37°C, and subsequently fixed with 4% formaldehyde for 30 min at 37°C and permeabilized with 0.1% Triton-X. Cells were imaged using a confocal microscope (Leica Microsystems GmbH; magnification, ×100) after counter-staining cells with DAPI (1 mg/ml) at room temperature for 5 min.
To detect the expression levels of IL-6 and IL-10 in CD68-positive cells, the sections were prepared as described for immunohistochemistry and incubated at 4°C overnight with mouse anti-CD68 (1:200; cat. no. ab955; Abcam), rabbit anti-IL-10 (1:200; cat. no. ab34843; Abcam;) or rabbit anti-IL-6 (1:200; cat. no. ab9324; Abcam) and goat anti-mouse IgG (H+L) unconjugated or goat anti-rabbit IgG (H+L) unconjugated secondary antibodies (1:5,000; cat. nos. BS13271 and BS12471; Biogot Technology Co., Ltd.) for 2 h at 37°C. Cells were counter-stained with DAPI (100 ng/ml) for 5 min at room temperature. Co-localization of CD68 with IL-6 or IL-10 was determined using an Olympus fluorescence microscope (magnification, ×100).

After the mice were sacrificed under deep anesthesia with pentobarbital sodium (1% in NS, 50 mg/kg, i.p.), the lumbar spinal cord segments were quickly extracted and stored in liquid nitrogen. The collected tissue samples were weighed, homogenized in lysis buffer, and centrifuged. Then the supernatant was collected. Protein samples were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto the polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Subsequently, the membranes were blocked with 5% nonfat milk for 2 h at room temperature and incubated with the primary antibodies: rabbit anti-iNOS (1:500, Abcam, Cambridge, UK), rabbit anti-arginase I (1:600, Santa Cruz, CA), rabbit anti-IL-1β (1:700, Abcam, Cambridge, UK), and rabbit anti-IL-10 (1:500, Abcam, Cambridge, UK) at 4°C overnight. The membranes were then washed with TBST and incubated for 1.5 h at room temperature with peroxidase affinipure goat anti-rabbit secondary antibody (1:10000, Jackson ImmunoResearch Inc, West Grove, USA). Then, the membranes were visualized using a chemiluminescent HRP substrate (Millipore Corporation, MA, USA) in an automated chemiluminescence system (Tanon, Shanghai, China). β-actin was used as a loading control for total protein, and ImageJ software (NIH, Bethesda, MD, USA) was used to measure the gray value of each band.