Odyssey infrared imager system 2

Retinas were prepared for Western blot analysis (n ¼ 3-4/ group) as previously described. 20 To isolate proteins, the frozen retinas were mechanically homogenized in 150 lL of lysis buffer (Radioimmunoprecipitation assay buffer; Cell Signaling Technology, Danvers, MA, USA) and a protease inhibitor (Sigma-Aldrich Corp., St. Louis, MO, USA). Ultrasound was then used for an accurate homogenization. Samples were placed on ice for 50 minutes, followed by centrifugation at 48C at 13,200 rpm for 30 minutes to reduce the last solid particles. The protein concentration was measured using a commercial bicinchoninic acid assay (BCA protein assay kit; Pierce Technology, Holmdel, NJ, USA). We applied 20 lg per lane to a 12% bis-Tris gel (NuPAGE; Invitrogen, Carlsbad, CA, USA) for electrophoresis and blotted afterward, using a transfer buffer (NuPAGE) for 60 minutes at 200 V. Nitrocellulose membranes were blocked in a solution of 5% milk powder/PBS/0.05% Tween-20. Primary antibodies were applied and incubated overnight, followed by secondary antibodies for 60 minutes (Table ). An Odyssey infrared imager system 2.1 (LI-COR Biosciences, Lincoln, NE, USA) recorded and analyzed the stained protein bands. Two measurements of each animal were used for statistical analysis.