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Fig. 1 | Acta Neuropathologica Communications

Fig. 1

From: Brainstem tau pathology in Alzheimer’s disease is characterized by increase of three repeat tau and independent of amyloid β

Fig. 1

CENSUS (Complete ENumeration and Sorting for Unlimited Sectors) via immunofluorescence to count all neurofibrillary changes and sort everything by a virtual slide system. Image acquisition by virtual slide system and subsequent quantitative analysis and ROI mapping. (a) Image capturing at the coordinated anatomical levels. Midbrain sections included the superior colliculus (SC) and red nucleus (RN), and the pontine sections included the locus coeruleus (LC) and the superior cerebellar peduncles. The snapshots captured with 10 times objective lens in separate fluorescence channels were put together to cover the entire tectum and tegmentum. 5 vertical planes at 1 μm intervals were simultaneously captured. (b) The extended focus imaging (EFI) module picked up pixels with maximum local contrast to make a single in-focus image from 5 vertical planes. (c-e) Representative parts of the separate channels of the extended-focused images and their overlays are shown. Bar = 20 μm. (f-h) These images were binarized by Triangle algorithm (f, g), followed by binary colocalization analysis on ImageJ (h). (i-n) Particle analysis program sorted all immunolabels by size, which we defined as neuroil threads (NTs, area 1–200 μm2, i-k) and neurofibrillary tangles (NFTs, area > 200 μm2, l-n), with retrospective inspection into the original counterpart for confirmation. Outlines are shown. (o-t) ROI mapping of the immunolabels. The outlines of the ROIs selected in area analysis are uniformly exaggerated and colored (4R tau/green, 3R tau/red, colocalization/yellow) on ImageJ to abstract the immunolabels of NTs (o-q) and NFTs (r-t), as automatic immunolabel mappings

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