Figure 4
From: Three-dimensional imaging and quantitative analysis in CLARITY processed breast cancer tissues
CLARITY allows for an unbiased pathology score based on Ki67 expression. (a) Schematic diagram depicting the methodology and optical slice selection used to compare individual 2D FFPE sections (5 µm, left) to the corresponding 3D thick section (500 µm, right) for Ki67 scoring. Blind scoring was done by two independent pathology professionals and results were analyzed with GraphPad Prism 7.0. (b) Images of tumor tissue #7 before sectioning, after sectioning, post clearing, and the corresponding FFPE H&E staining of the tissue (10x, 40x). Representative confocal images of tumor #7 (c) 2D FFPE immunofluorescence staining (25X) and (d) 3D volumetric (top row) and optical slices (bottom row) HM embedded thick tissue immunofluorescence staining (25X). Blue: DAPI, Green: pan-CK, Yellow: Ki67, Red: CD3, 25X (e) A comparison of Ki67 positive ratio (Ki67 positive epithelial/total epithelial) in corresponding FFPE thin sections and HM thick sections (p-values as listed, unpaired t test) in biopsy samples. (f) Representative images of tumor #8 with heterogenous expression of Ki67 in FFPE 2D thin sections from different sections (25X) demonstrating differing levels of Ki67 positive rates.
