Quantification of Hippocampal and Ventricle Volumes

Hippocampal brain sections (seven sections per mouse, 30-µm thick, 300 µm apart) were mounted onto microscope slides (Fisher Scientific). A 0.1% Sudan Black solution was prepared by adding Sudan Black powder (Sigma) to 70% ethanol (KOPTEC) and mixing the solution using a magnetic stirrer. The solution was then centrifuged at 1,100g for 10 min and the collected supernatant was filtered using a 0.2-µm filter syringe (Thermo Scientific). Sections were then stained with the 0.1% Sudan Black solution for 10 min and washed in 70% ethanol and then in Milli-Q water. Sections were coverslipped with ProLong Gold mounting medium (Invitrogen) and imaged on an Aperio VERSA slide scanning microscope (Leica) at ×10 magnification. To quantify the volumes of the hippocampus and posterior lateral ventricle, we traced the areas of interest in ImageJ and used the formula: volume = (sum of area) × 0.3 mm (ref. ^{28 (link)}). We took a sum of all seven brain sections per mouse, roughly between coordinates AP = −1.2 and AP = −3.4.