Apolipoprotein D Transgenic Mice Develop Hepatic Steatosis through Activation of PPAR? and Fatty Acid Uptake

Labrie, Marilyne; Lalonde, Simon; Najyb, Ouafa; Thiery, Maxime; Daneault, Caroline; Des Rosiers, Chrisitne; Rassart, Eric; Mounier, Catherine

Human apoD was quantified in plasma and liver homogenate of transgenic Tg-apoDH mice by Elisa. 96-well ELISA plates were coated overnight at 4°C with purified human apoD standards (with range concentration of 0-10 μg/mL) and samples (for plasma, 10 μL and for liver, 5 μg) diluted in 0.1M sodium carbonate pH 9.5 to a final volume of 100 μL. The coated wells were blocked with 3% BSA for 1h at RT and were incubated overnight at 4°C with an antibody against biotinylated human apoD (1:10000, biotinylated H-apoD antibody (43) ). After washing, the wells were treated with HRP-streptavidin (1:25000) for 1h at RT. After washing, peroxidase substrate TMB (3,3', 5,5'-Tetramethylbenzidine) solution (100 μL, Fitzgerald, MA, USA) was applied to each well for 30 min and the reaction was stopped by adding 50 μL of 1M phosphoric acid. Absorbance values (at 450 nm) were obtained with Elisa Plate Reader (Tecan Infinite M1000, Tecan US, NC, USA).