Anti rabbit or mouse igg alkaline phosphatase secondary antibodies

For the analysis of total 3-nitrotyrosine (3-NT) and 4-hydroxy-2-nonenal (HNE)-bound protein levels, 10 μl of hippocampus homogenate were incubated with 10 μl of Laemmli buffer containing 0.125 M Tris base pH 6.8, 4% (v/v) SDS, and 20% (v/v) glycerol. The resulting samples (250 ng per well) were loaded onto a nitrocellulose membrane with a slot-blot apparatus under vacuum pressure. The membrane was blocked for 2 h with a solution of 3% (w/v) bovine serum albumin in PBS containing 0.01% (w/v) sodium azide and 0.2% (v/v) Tween 20 and incubated respectively with primary antibodies anti-HNE (Alpha diagnostic, San Antonio, TX, USA) and anti-3NT (Santa Cruz Biotechnology, Dallas, TX, USA) for 2 h at RT. Membranes were washed and incubated with anti-rabbit or mouse IgG alkaline phosphatase secondary antibodies (Sigma-Aldrich, St Louis, MO, USA) for 1 h at room temperature. The membrane was developed with Sigma fast tablets (5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium substrate [BCIP/NBT substrate], Sigma-Aldrich, St Louis, MO, USA). Membranes were dried and the image was acquired using ChemiDoc XP image system and analyzed using Image Lab software (Bio-Rad Laboratories, Hercules, CA, USA).