Horseradish peroxidase conjugated antibody and enhanced chemiluminescence

Washed cultured cells and retinal tissue samples were lysed in modified RIPA buffer supplemented with 1:100 (v/v) of proteinase/phosphatase inhibitor cocktail (Thermo Scientific, Greenville, SC, USA). This was followed by centrifugation at 12,000 xg at 4°C for 30 min to remove insoluble material. Protein was determined by BCA Protein Assay (Thermo Scientific, Greenville, SC, USA) and equal amount of protein was exposed to boiling in Laemmli sample buffer, separated by SDS-PAGE on a gradient gel (4 to 20%, Pierce, Rockford, IL), then transferred to nitrocellulose membrane, followed by incubation with specific antibodies. Antibodies for NMDAR1 (Cell signaling, Danvers, MA, USA, Cat# 5704S), NMDAR2A (Cell signaling, Danvers, MA, USA, Cat# 4205s), NMDAR2B (Cell signaling, Danvers, MA, USA, Cat# 4207s), ZO-1 (Abcam, Cambridge, MA, USA, ab59720), occludin (Invitrogen, Waltham, MA, USA, 71-1500), Albumin (Bethyl, TX, USA), GAPDH, (Sigma-Aldrich, St. Louis, MO, USA) and β actin (Cell signaling, Danvers, MA, USA, Cat# 937215) were detected with a horseradish peroxidase-conjugated antibody and enhanced chemiluminescence (Thermo Scientific, Greenville, SC, USA). Intensity of immunoreactivity was measured by densitometry using Image J software (NIH).