In order to qualitatively assess levels of pTau expression in 3xTg-AD mice 1month post alcohol or saccharin intake (Fig. 3), a subset of mice (N = 2) were deeply anesthetized with 100mg/kg pentobarbital and were intracardially perfused with freshly prepared, ice cold, phosphate buffered saline (1M PBS, pH 7.4), followed by 4% paraformaldehyde (PFA). Whole brains were extracted, post-fixed in 4% PFA for 48h, and then stored in PBS at 4°C. Coronal brain sections were sliced using a vibratome (Leica VT1000S) and stored in cryoprotectant (recipe) at −20°C until IHC was conducted. A subset of coronal brain sections from the anterior HPC and AMY of WT and 3xTg-AD mice with a history of saccharin or alcohol drinking were processed for fluorescent immunohistochemistry as previously published (Besheer & Hodge, 2005; Salling et al., 2016; Stevenson et al., 2009). Briefly, sections were incubated in methanol to facilitate cell permeabilization, in hydrogen peroxide to block endogenous peroxidase activity and in a blocking buffer of Normal Goat Serum (Vector Labs) +0.1%Triton-X in PBS (PBSTx) to inhibit non-specific binding. Next, sections were incubated overnight at
