Disposable tissue grinders

Frozen brain tissue samples from two different MSA subjects and one age-matched control subject were obtained from UCLA Brain Tumor Translational Resource (BTTR) and The Human Brain and Spinal Fluid Resource Center (NIH Neurobiobank). From the frozen brain tissue samples we extracted insoluble protein aggregates using previously published protocol that included precipitation with Phosphotungstate anion (PTA) and the ionic detergent sarkosyl (Woerman et al., 2015 (link); Prusiner et al., 2015 (link)). Briefly, frozen tissue from each subject was homogenized in ice cold PBS (10% wt/vol) using Fisherbrand Disposable Tissue Grinders (Cat # 02-542-10). The brain homogenate was centrifuged at 1500 x g for five mins. The supernatant was combined with benzonase and sarkosyl to a final concentration of 0.5% and 2%, respectively. The mixture was incubated at 37°C in a Torrey Pine shaker at speed 9 for 2 hr. PTA, pH 7.0 was then added to a final concentration of 2%. This mixture was incubated at 37°C in a Torrey Pine shaker at speed 9 overnight. The sample was then centrifuged at 16000 x g for 30 min at room temperature. The resulting pellet was re-suspended in 2% sarkosyl and 2% PTA solution and incubated for 1 hr. The sample was centrifuged at 16000 x g for 30 min at room temperature. The resulting pellet was re-suspended in 10% of the initial starting volume of PBS.