Rabbit anti irisin antibody

Lung tissues were washed twice with ice-cold PBS and lysed in lysis buffer [10mM tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 1mM phenylmethylsulfonyl fluoride, and leupeptin and aprotinin (10 mg/ml each)]. After centrifugation at 12,000g for 15 min, the supernatants were collected and their protein concentrations were measured using a BCA protein assay kit (HyClone Pierce). The tissue homogenates (50 μg of protein) were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. The blots were then washed with tris-buffered saline with Tween 20 (TBST), blocked with 5% milk powder in TBST buffer for 1 hour, and incubated with the appropriate primary antibodies at appropriate dilutions. The blotted membranes were probed with the rabbit anti-irisin antibody (1:500; Phoenix Pharmaceuticals), the rabbit anti-UCP2 antibody (1:500; Sigma-Aldrich), rabbit anti-active caspase 9 antibody (1:500; Abcam), and rabbit anti–cytochrome c antibody (1:500, Abcam) at 4°C overnight. Then, the membranes were washed and primary antibodies were detected with fluorescent-labeled goat anti-rabbit IgG (1:15,000; IRDye, LI-COR), and the bands were visualized by Odyssey Western Blot Detection System (LI-COR). The amount of protein transferred onto the membranes was verified by immunoblotting for glyceraldehyde-3-phosphate dehydrogenase.

Lung tissues, cleared of blood with phosphate-buffered saline (PBS) and kept in 4% paraformaldehyde for 1 to 2 days at 4°C, were embedded in paraffin, sectioned, and mounted on slides. After deparaffinizing and rehydrating by xylene and different concentrations of ethanol, the sections were stained with hematoxylin solution for 10 min, washed with running water for 5 min, soaked in 1% acid alcohol for 1 min, and counter-stained with eosin Y solution for 1 min; alternatively, the sections were treated with 0.1% Triton X-100 and 10 mM sodium citrate (pH 6.0) and blocked with 5% goat serum in PBS for 1 hour at 37°C for immunohistochemistry. The tissues were incubated with rabbit anti-irisin antibody (1:200; Phoenix Pharmaceuticals) overnight at 4°C.