Objective: Human brain has features with high fat, high density, difficulty in deep and even labeling, and strong background fluorescence, which made it uneasy to get high-resolution imaging. To solve the above problems, this study successfully explored a new method for human brain transparency called dFC-Tesos by steps including human brain tissue electrophoresis degrease, antibody labeling, decolorization, transparency, three-dimensional high-resolution imaging, to clearly observe the spatial relationships and pathological changes between microglia and Aβ plaques in Alzheimer's disease.
Methods: The 3 mm thick slices of the superior temporal gyrus of the human brain were sectioned by a vibration slicer, then degreased in 4% SDS solution with 3 V, 0.01 A electric field until the brain slices were lightly permeable and the interface between gray matter and white matter was fuzzy. Antibodies of microglia were incubated and Aβ plaques were stained by thioflavin S. Additionally, to exclude endogenous fluorescence interference of the samples, the brain slices were decolorized by 8% ammonia, hydrogen peroxide containing 0.08% KOH and 25% N,N,N,N-tetra(2-hydroxypropyl) ethylenediamine (w/v), respectively, which successfully reduced the interference of vascular auto-fluorescence and improved the imaging quality. Human brain tissue transparency was accomplished by tert-butanol gradient degreasing, tert-butanol, and tetraethylenediamine dehydration, benzyl benzoate, bisphenol A ethoxylate diacrylate and tetraethylenediamine for refractive index matching. They were then imaged by LS18 lightsheet micro-imaging instrument with a resolution of 1.65 μm × 1.65 μm × 3.5 μm.
Results dFC-Tesos method was sequentially performed by electrophoretic degreasing of human brain samples, electrophoretic antibody labeling, decolorization, transparent human brain sample preparation, light-sheet microscopy, and splicing of big data to successfully obtain ultrastructural information and spatial location relationships of microglia and Aβ plaques in Alzheimer's disease (AD) brain, which provided methodological support for the study of new mechanisms of the pathology of AD. The dFC-Tesos method also provided an important tool for the study of macroscopic and ultrastructural structures of the pathological brain, the mechanism of major brain diseases, as well as the study of drug targets, and so on.
