Complete mammalian protease inhibitor

To quantitatively extract soluble Aβ from TgSwDI mice brain, 150 mg of brain tissue was polytron homogenized in diethylamine (DEA) buffer (50 mM NaCl and 0.2% DEA), with complete mammalian protease inhibitor (Sigma-Aldrich) and centrifuged at 21,000g for 45 min at 4°C. Supernatants were collected and analyzed for Aβ₄₀ and Aβ₄₂ brain levels using commercially available ELISA kits according to the manufacturer instructions (ThermoFisher). All samples were run at least in triplicates and corrected to the total protein amount in each sample using BCA assay.

To quantitatively extract total Aβ from the brain tissues, a three-step serial extraction procedure was used as descried previously [29 (link)]. Briefly, 150 mg of brain tissue was polytron homogenized in DEA buffer (50 mM NaCl, 0.2% diethylamine, with complete mammalian protease inhibitor (Sigma-Aldrich, MO) and centrifuged at 21,000×g for 45 min at 4°C. Supernatant (DEA fraction) was collected and the pellet was re-extracted with 0.5 ml of 2% sodium dodecylphosphate (SDS; Sigma-Aldrich) and centrifuged as described above. Again, the supernatant (SDS fraction) was collected and pellet was re-extracted with 0.5 ml of 70% formic acid (FA) and centrifuged for 2 h at 21,000×g at 4°C. The supernatant (FA fraction) was collected without disrupting the pellet or floating lipid layer. All fractions were stored at −80°C until the time of analysis. The total amount of Aβ in the brain was calculated by summing the amount of Aβ in all fractions.