Mouse anti cd86

Rabbit anti-α-SMA (Cat No. ab179467), Col4α1 (Cat No. ab6586), CD206 (Cat No. ab64693), CD68 (Cat No. ab125212), PPARα (Cat No. ab227074), and mouse anti-CD86 (Cat No. ab220188) antibodies were purchased from Abcam (Cambridge, MA). Rabbit anti-F4/80 (Cat No. #30325), NF-κB-p65 (Cat No. #8242), and phosphorylation of NF-κB-p65 (Cat No. #3033) antibodies were purchased from Cell Signaling Technology. Rabbit anti-β−actin (Cat No. AC028) was purchased from ABclonal (Wuhan, China). Mouse AST ELISA kit (Cat No. ab263882), ALT assay kit (Cat No. ab241035), ALP assay Kit (Cat No. ab267583), gamma-glutamyl transferase (γ-GT) assay kit (Cat No. ab241029), triglyceride assay kit (Cat No. ab65336), free fatty acid assay (Cat No. ab65341), ROS detection assay (Cat No. ab139476), caspase-3 assay kit (Cat No. ab39401), and mitochondrial complex I enzyme activity microplate assay kit (Cat No. ab109721) and mitochondrial complex III activity assay kit (Cat No. ab287844) were obtained from Abcam (Cambridge, MA). Glycyrrhetinic acid (Cat No. HY-N0375), betaine (Cat No. HY-B0710), ursolic acid (Cat No. HY-N0140), and wogonin (Cat No. HY-N0400) were purchased from MedChemExpress (Shanghai, China).

To determine whether exercise intervention could affect macrophage polarization in PVAT, the paraffin-embedded PVAT sections (10 μm thickness) were deparaffinized and rehydrated prior to antigen unmasking by boiling in 10 mM sodium citrate (pH 6.0) for 10 mins. Sections were cool down for 30 mins, then washed with distilled water for twice, 5 mins of each. After permeabilization with permeabilization solution (3% BSA with 0.1% triton-100 in PBS) for 1 hr, sections were incubated with primary antibodies rat anti-F4/80 (Abcam, 8 μg/ml), mouse anti-CD86 (Abcam, 4 μg/ml), or rabbit anti-CD206 (Abcam, 4 μg/ml) for overnight at 4°C. Followed by three times wash with PBS, sections were probed with FITC- or Cy3-conjugated secondary antibody for 2 hrs at RT. Cell nucleus was count stained with DAPI for 5 mins at room temperature. Images were taken under a fluorescence microscope (Nikon, Eclipse E600). F4/80+CD86+ cells were considered as M1 macrophages, and F4/80+CD206+ cells were considered as M2 macrophages. Cell numbers were averaged from 5 different fields. Data from ten individual sections represented the data from one mouse.