Immunohistochemistry

Fixed hemibrains were sectioned exhaustively in the horizontal plane at 40 μm using a vibratome (Leica Biosystems). Sections were stored singly in PBS with 0.03% sodium azide at 4°C until immunohistochemistry was performed. Every eighth section (from a total of ∼100 per brain) was immunostained for β-amyloid (Aβ) as previously described (Christensen and Pike, 2017). In brief, tissue sections containing hippocampus were pretreated with 95% formic acid for 5 min, then washed three times for 5 min in TBS, followed by a 10 min rinse with an endogenous peroxidase blocking solution. Next, sections were rinsed in TBS/0.1% Triton-X before being incubated for 30 min in a blocking solution consisting of TBS/2% bovine serum albumin. Sections were incubated overnight at 4°C with anti-Aβ antibody (1:300; Life Technologies; Cat #71-5800) diluted in blocking solution. Immunostaining was also conducted in the absence of formic acid pretreatment with the following primary antibodies: doublecortin (1:2500; Santa Cruz) as a marker of new neurons, clone AT8 (1:750; Thermo) for phosphorylated tau (phospho-tau), Iba-1 (1:2000; Wako) for microglia. Sections incubated in primary antibody were washed and then incubated with the appropriate secondary antibody (Vector Laboratories) for 1 h and processed for diaminobenzidene visualization using Vectastain ABC Elite kit (Vector Laboratories). Stained sections were air-dried overnight, dehydrated in a series of graded alcohols, then coverslipped with Krystalon (EMD Millipore).