Oxiselect 4 hne adduct competitive elisa kit

Retinas were isolated under a microscope from WT mice and Glast^± mice treated with vehicle or AST at the end of 5 weeks of age. Protein concentration and 4-HNE levels in the retina were determined by BCA protein assay (Thermo Fisher Scientific, Waltham, MA) and the OxiSelect 4-HNE adduct competitive ELISA kit (Cell Biolabs, City, Country), respectively. Eight eyes from four animals were used for each group.

Six (n = 5) and 24 h (n = 6) after SCI with 30 g/mm² of clipping power for a duration of 15 s, the spinal cord was dissected into rostral, epicentre, and caudal segments, each of which was 2 mm in length. Each spinal cord segment was minced with a homogenizer in RIPA buffer containing 50-mM Tris–HCL 7.5 (Nippon Gene Co., Ltd), 150-mM NaCl (Nacalai Tesque), 0.1% SDS (Nippon Gene Co., Ltd), 1% NP-40 (Sigma-Aldrich), 5-mM EDTA with pH 8.0 (Nippon Gene Co., Ltd ), and 1 mM PMSF (Wako). Protease inhibitor cocktail (Thermo Fisher Scientific) was added to the buffer prior to homogenization in accordance with the manufacturer’s instructions (1:100). The spinal cord was homogenized for ∼40 grinds using constant force to ensure consistency and homogeneity of the samples. The homogenates were divided into roughly equal volumes in Eppendorf tubes and stored at 4°C for 30 min. The homogenates were then centrifuged at 13 000 rpm for 20 min at 4°C. The resulting supernatant was decanted, placed into newly labelled Eppendorf tubes, and immediately stored at −80°C. The protein concentration was determined using Qubit (Thermo Fisher Scientific). Equal amounts of protein were examined. Lipid peroxidation was quantified as a marker of oxidative stress using the OxiSelect 4-HNE Adduct Competitive ELISA Kit (Cell Biolabs, Inc., San Diego, CA, USA).

Oxidative stress was measured by OxiSelect™ 4-HNE adduct competitive ELISA kit (Cell Biolabs, San Diego, CA) according to the instructions. Whole cell lysate (50 µg protein equivalent) for each sample was used for the assay.