Quantifying Amyloid-Beta in Brain Tissues

Aβ ELISA was performed as previously described [23, 24] (Liu et al., 2014 (link); Fang et al., 2016 (link)). Briefly, cortical and hippocampal tissues were homogenized in Tris-buffered saline (TBS) containing a protease inhibitor cocktail (Sigma) and then centrifuged at 16,000 ×g for 30 min at 4°C. The supernatant (TBS-soluble fraction) was collected, and the pellets were homogenized in TBS supplemented with 1% Triton X-100 (TBST) containing a protease inhibitor cocktail (Sigma), sonicated for 5 min at 4°C in a water bath, and then centrifuged at 16,000 ×g for another 30 min at 4°C. The supernatant (TBST-soluble fraction) was collected, and the pellets were extracted a third time with an ice-cold guanidine buffer (5 M guanidine HCl/50 mM Tris, pH 8.0) and referred to as the guanidine-soluble fraction. The protein concentration was determined using a bicinchoninic acid protein assay kit (Beyotime Biotechnology). The Aβ concentrations were detected by Aβ40 and Aβ42 ELISA kits (Invitrogen) following the manufacturer’s instructions. The levels of Aβ were normalized to the amount of total protein in the tissues.