Anti ho 1 antibody

Cortical brains of the sham and HS rats were collected. Tissues were lysed in a buffer containing 150 mM NaCl, 20 mM Tris-HCl (pH 7.4), 1% sodium deoxycholate, 0.1% SDS, 1% NP-40, and 10% complete protease inhibitor solution (Roche Molecular Biochemicals, Mannheim, Germany). Twenty micrograms of protein lysate were run on 4%-12% NuPAGE Bis-Tris polyacrylamide gels (Invitrogen). Proteins were transferred from the gels to PVDF membranes (Invitrogen, USA) by electroblotting. Each membrane was incubated in a blocking buffer containing phosphate-buffered saline (PBS; 120 mM NaCl, 2.7 mM KCl, and 0.01 M PB), 3% BSA (Sigma, St. Louis, MO, USA), and 0.04% Tween 20 for at least 1 h. Each membrane was then blocked, incubated with an anti-HO-1 antibody (1:2000; Stressgen Biotechnologies, Victoria, British Columbia, Canada) at 4°C overnight, washed in PBS containing 0.04% Tween 20 (three times, 10 min each) to remove unbound primary antibodies, incubated with an HRP-conjugated secondary antibody (1:10,000 diluted in blocking buffer, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 2 h, and washed in PBS (three times, 10 min each). Labeled proteins were visualized by chemiluminescence (ECL Plus kit, Amersham, Arlington Heights, IL, USA).