Lipid and Amyloid Analysis in E4FAD Mice

Serial sagittal sections (35 μm thick and separated by 280 μm) from E4FAD mouse brains were used for staining/immunostaining measures. For lipid staining, the sections were washed in TBS, mounted on glass microscope slides, and dried for 1 h. In the dark, 1X LipidSpot-610, “a fluorogenic neutral lipid stain that rapidly accumulates in lipid droplets where it becomes brightly fluorescent” (Biotium product sheet), was applied directly to the slides and incubated for 1 h (previously described [95 (link)]). The stained sections were imaged at 63X with a Zeiss Fluorescent Microscope, one field for subiculum (SB) and three fields for CX: visual CX, somatosensory CX, and frontal CX. Images were analyzed by counting the number of LD per nuclei (10–14 nuclei per field) and for the CX, averaged per mouse. For amyloid deposition, the sections were washed in TBS and stained with Thio-S. For Aβ deposition, astrogliosis, and microgliosis, sections were immunostained using MOAB-2, GFAP, and Iba1 antibodies, respectively (previously described [13 (link), 96 (link)]). Whole sections were imaged at 10X magnification with a Zeiss Fluorescence microscope and analyzed for area covered by Thio-S, Aβ, GFAP, and Iba-1 in CX using ImageJ software.