Arc0102

A total of 8 slides for each group were then incubated with primary antibodies: TNF-α (1:100; NBP1-47581, Novus, USA), HIF-1α (1:100; NB100-479, Novus, USA), IL-10 (1:100; ARC0102, Invitrogen, USA), and ghrelin (1:100; NBP1-51224, Novus, USA) for 1 h at 37 °C. The slides were then incubated with the secondary antibody (859043 Histostain-Plus Kit, Invitrogen, USA) at 37 °C for 30 min, washed with a phosphate-buffered saline buffer, and 3-amino-9-ethylcarbazole (AEC) (2007, AEC reagent set, Invitrogen) staining was applied. The sections were counterstained with Mayer's hematoxylin (MHS16, Sigma-Aldrich, USA) (9) . All sections were photographed using an Olympus C-5050 digital camera. For each specimen, a score was assigned according to the percentage of positive cells: <5% of the cells: 1 point; 6-35% of the cells: 2 points; 36-70% of the cells: 3 points; and >71% of the cells: 4 points. The percentage indicated how many cells out of 100 had stained positive for the targeted protein per area. The intensity of staining was also quantified, with negative staining equal to 1 point; weak staining, 2 points; moderate staining, 3 points; and strong staining, 4 points. The expression score was then calculated by multiplying the two scores above (23) .