Full- versus Sub-Regional Quantification of Amyloid-Beta Load on Mouse Brain Sections

Abstract

Extracellular accumulation of amyloid-beta (Aβ) plaques is one of the major pathological hallmarks of Alzheimer's disease (AD), and is the target of the only FDA-approved disease-modifying treatment for AD. Accordingly, the use of transgenic mouse models that overexpress the amyloid precursor protein and thereby accumulate cerebral Aβ plaques are widely used to model human AD in mice.Therefore, immunoassays, including enzyme-linked immunosorbent assay (ELISA) and immunostaining, commonly measure the Aβ load in brain tissues derived from AD transgenic mice. Though the methods for Aβ detection and quantification have been well established and documented, the impact of the size of the region of interest selected in the brain tissue on Aβ load measurements following immunostaining has not been reported. Therefore, the current protocol aimed to compare the Aβ load measurements across the full-and sub-regions of interest using an image analysis software. The steps involved in brain tissue preparation, free-floating brain section immunostaining, imaging, and quantification of Aβ load in full-versus sub-regions of interest are described using brain sections derived from 13-month-old APP/PS1 double transgenic male mice. The current protocol and the results provide valuable information about the impact of the size of the region of interest on Aβ-positive area quantification, and show a strong correlation between the Aβ-positive area obtained using the full-and sub-regions of interest analyses for brain sections derived from 13month-old male APP/PS1 mice that show widespread Aβ deposition.

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“…Slides were then sealed with the Vectamount aqueous mounting media (Vector Laboratories, Newark, CA, USA) and stored at 4 °C until imaging. The Thio-S- or 6E10-stained brain tissue sections were imaged using a BZ-X710 Keyence fluorescence microscope (Keyence, Itasca, IL, USA) under a 2 × objective to capture the entire brain tissue section in one image, and digitized images were analyzed using the NIH ImageJ software (version 1.53e), National Institutes of Health, Bethesda, MD, USA ( ) as published previously [ 22 ].…”

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“…Immunofluorescence of brain sections was viewed and captured with the Zeiss Axio imager.M2m microscope (Axiovision 4.8) and processed by ImageJ software (v 1.53e). The immunoreactive areas were quantified using ImageJ as previously described 10 . The average data of at least three sections per mouse was used to reduce the variance among tissue sections.…”