Isolation of Soluble and Insoluble Protein Fractions

To isolate protein for the ELISA, flash-frozen brain hemispheres were microdissected into cortical, hippocampal, and thalamic regions and grounded to a powder. Hippocampal tissue was then homogenized in Tissue Protein Extraction Reagent (TPER (Life Technologies, Grand Island, NY)) with protease and phosphatase inhibitors present. Samples were centrifuged at 100,000 g for 1 h at 4 °C to generate TPER-soluble fractions. To generate formic acid fractions, protein pellets from the TPER-soluble fraction were then homogenized in 70% formic acid and centrifuged at 100,000 g for 1 h at 4 °C, the formic acid fraction is then neutralized. Quantification of soluble and insoluble fractions of both Aβ and NfL was performed as previously described [67 (link)].