Sudan black powder

Serial coronal hippocampal brain sections (7 sections per mouse, 30 μm thick, 300 μm apart) were mounted onto microscope slides (Fisher Scientific) and dried at room temperature for 1 h. The 0.1% Sudan Black solution was prepared by adding the appropriate amount of Sudan Black powder (Sigma) to 70% ethanol (KOPTEC) and mixing the solution using a magnetic stirrer while and protected from light. The solution was then centrifuged at 3,000 RPM for 10 min and the collected supernatant was filtered using a 0.2 μm filter syringe (Thermo Scientific) to remove undissolved dye. Sections were then stained with the 0.1% Sudan Black solution at room temperature for 10 min and washed 3×2min in 70% ethanol and 3×5min in Milli-Q water. Sections were then coverslipped with ProLong Gold mounting media (Invitrogen) and imaged on an Aperio VERSA slide scanning microscope (Leica) at 10X magnification. For hippocampal and posterior lateral ventricle volumetric analyses, the areas of interest were traced in ImageJ using the segmented line tool and the volume was calculated using the formula: volume = (sum of area) * 0.3 μm^{22 (link)}. The sum of area value was obtained by taking a sum of the quantified area measurements of all 7 brain sections per mouse, roughly between coordinates AP=−1.2 and AP=−3.4.

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.