Plates were stained at appropriate selected times with crystal violet and quantified at 570 nm upon dissolution in Sorenson's buffer.
The resulting extract is fractionated by HPLC on a column of silica gel, to separate the MDA-TBA adduct, which finally is eluted with methanol/phosphate buffer and quantified spectrophotometrically at 532 nm.
Primary antibodies were detected by incubation with appropriate IRDye secondary antibodies (LI-COR Biosciences) for 1 hour at RT in Odyssey blocking buffer and quantified using the Odyssey infrared imaging system (LI-COR Biosciences).
Primary antibodies were detected by incubation with appropriate IRDye® secondary antibodies (LI-COR Biosciences) for 1 h at room temperature in Odyssey® blocking buffer and quantified using the Odyssey® infrared imaging system (LI-COR Biosciences).
The isolated DNA was finally suspended in 100 µl of elution buffer and quantified on 1 % agarose gel.
Fibronectin remaining after the extraction was then solubilized with SDS buffer and quantified by immunoblotting as described above.
Proteins were extracted by incubation with RIPA buffer and quantified by Bradford reagent.
Protein was extracted from BAT using RIPA buffer and quantified by the Bio-Rad DC protein assay.
The oligonucleotides were dissolved in the CMG buffer and quantified by A260.
After 1 h incubations at room temperature, the reactions were quenched using 4 m hydroxylamine and the samples were purified, eluted in 35 μL of aaRNA elution buffer and quantified using a NanoDrop Spectrophotometer ND-1000 (NanodroProductsts, Wilmington, DE, USA).
