Quantifying Brain Amyloid Binding

Unfractionated whole brain tissue homogenates (described above) were diluted from 300 mg/ml to a concentration 10 mg/ml in PBS prior to the binding assay as previously described (Ikonomovic et al., 2008 (link)) with the exception of the fold-higher initial homogenate prepared in the current study. For determination of ³H-PiB binding, 1 nM ³H-PiB (American Radiolabeled Chemicals, St. Louis, MO, USA; specific activity 72.4 Ci/mmol) was incubated with 100 μg tissue in 1 ml PBS as described previously (Ikonomovic et al., 2008 (link)). Unlabeled PiB was dissolved in DMSO at 400 mM (to yield 51% DMSO) and this stock solution was diluted with PBS to achieve the desired concentration for the binding assay. Non-specific binding was defined as the number of counts remaining in the presence of 1 mM unlabeled PiB. The binding mixtures were filtered through a Whatman GF/B glass filter using a Brandel M24R cell harvester (Brandel, Gaithersburg, MD) and rapidly washed five times with 3 ml PBS. The filters were counted in Cytoscint-ES after thorough vortex mixing and resting overnight. Results were corrected for non-specific, non-displaceable binding in the presence of 1 mM PiB and expressed as picomoles ³H-PiB bound per gram of wet brain tissue weight in the homogenate.