Azure biosystem c500 machine

After appropriate treatments and rinsing with cold phosphate-buffered saline, REC were collected in lysis buffer containing protease/phosphatase inhibitors and scraped into their respective tubes. Retinal extracts were prepared by sonication. Equal amounts of protein from cell or tissue extracts were separated on pre-cast tris-glycine gels (Invitrogen, Carlsbad, CA) and blotted onto a nitrocellulose membrane. After blocking in TBST (10mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and 5% (w/v) BSA, membranes were treated with the following primary antibodies: TLR4, IL-1 receptor-associated kinase 1 (IRAK1), TNF receptor-associated factor 6 (TRAF6), MyD88, HMGB1 (Abcam, San Francisco, CA), NF-κB, phospho-NF-κB (Cell Signaling, Danvers, MA), and β-actin (Santa Cruz, Santa Cruz, CA); then followed by incubation with secondary antibodies (Fisher Scientific, Pittsburgh, PA) labeled with horseradish peroxidase. Antigen-antibody complexes were detected using a chemilluminescence reagent kit (Thermo Scientific, Pittsburgh, PA). Western blot images were collected on an Azure Biosystem C500 machine (Azure Biosystems, Dublin, CA). Mean densitometry of bands for each protein were measured.