Oxiselect mda adduct competitive elisa kit

Serum concentrations of BAFF and resistin were determined using ELISA (MBLYS0 for BAFF and MRSN00 for resistin; R&D Systems, Minneapolis, MN, USA). The MDA adduct levels in the liver and EAT were estimated using a commercially available ELISA kit (Oxiselect MDA Adduct Competitive ELISA kit; Cell Biolabs, San Diego, CA, USA), according to the manufacturer’s protocol.

Acute sagittal brain slices were prepared as described in the previous section and were allowed to recover in a vapor interface holding chamber (Scientific Systems Inc, Canada) containing standard ACSF at 34 C for 1 h. Slices were then assigned in an alternating pattern to either the control or the Aβ group, so that each slice incubated in Aβ-containing ACSF had its immediate neighbor from same hemisphere incubated in control ACSF. Following 1-h incubation in their respective oxygenated ACSF solutions, slices were snap-frozen over dry ice. Slices were then homogenized using a 26-gauge needle in the presence of a high-detergent buffer consisting of 50 mM Tris, 150 mM sodium chloride, 2% Nonidet P-40, 1% sodium deoxycholate, 4% sodium dodecyl sulfate, and supplemented with complete protease inhibitor cocktail (Roche), phosphatase inhibitor cocktail 1 (P2850, Sigma), phosphatase inhibitor cocktail 2 (P5726, Sigma), and 0.005% Butylated hydroxytoluene. The total protein in cell lysates was measured with the BCA protein assay kit (#23227, Pierce). Malondialdehyde (MDA) levels were quantified using a OxiSelect™ MDA Adduct Competitive ELISA Kit (Cell Biolabs, Inc, USA) as per manufacturer instructions. MDA levels were normalized to each sample’s protein concentration and the result then normalized to the average of all control slices from same hemisphere.

MDA assay was performed in order to measure the lipid peroxidation as a marker of oxidative stress, by using commercially available OxiSelect™ MDA Adduct Competitive ELISA Kit (Cell Biolabs, Inc., San Diego, CA, USA) by following the manufacturer’s protocol. Briefly, samples were added on the MDA conjugate-coated wells for 10 min. Subsequently, samples were incubated with anti-MDA antibody for 1 h, at room temperature. Following three washes, the secondary antibody was added for 1 h. Then, samples were incubated with the substrate solution from 2 to 20 min. The reaction was stopped by using stop solution and the absorbance at 450 nm was read using a microplate reader. For each sample the absorbance was adjusted for mg of proteins loaded.