Isolation and Analysis of Insoluble Tau

Slice cultures for assessment of insoluble tau from three wells (nine slices in total) were harvested into ice-cold PBS and presented as n = 1. Slices were washed in PBS, pelleted, and sarkosyl extractions were performed as previously described [29 (link)]. Slice cultures for comparison of lysate purified and unpurified media preparations from three wells (nine slices in total) were harvested into ice-cold PBS and presented as n = 1. Slices were lysed in radio immunoprecipitation assay buffer containing 1% triton X-100 (Boston Bioproducts) and a cocktail of protease inhibitors (Sigma-Aldrich). Lysates were centrifuged at 1000 x g and soluble supernatants were subjected to western blot analysis.