V120 virtual slide microscope

Frozen tissue sections, 7 μm in thickness, were fixed in a mixed solution of acetone (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan): chloroform (FUJIFILM Wako Pure Chemical Corporation) at 1:1 ratio for 10 min at 4 °C. Next, the sections were air-dried and incubated in a staining solution prepared by mixing 8 mg glycyl-prolyl-4-methoxy-β-naphthylamide hydrochloride (Sigma-Aldrich, Saint Louis, MO, USA) dissolved in 1 mL dimethyl sulfoxide (FUJIFILM Wako Pure Chemical Corporation) and 1 mg Fast Blue BB Salt hemi zinc chloride salt (Sigma-Aldrich) dissolved in 1 mL PBS (pH = 7.0) at a 1:20 ratio. After incubation for 20 min at room temperature, the tissue sections were incubated for 5 min in 2% CuSO₄ and rinsed with MQ and fixed with 10% formalin. After washing with MQ, the sections were counterstained with Carazzi’s hematoxylin (Muto Pure Chemicals, Tokyo, Japan). Images were acquired using a V120 Virtual Slide microscope (Olympus, Tokyo, Japan). Areas positive for DPPIV were quantified using ImageJ.

Whole sections were scanned at a 20x objective magnification using a V120 virtual slide microscope (Olympus, Tokyo, Japan). Thirty regions of interest (ROIs) were selected in a zigzag sequence along the cortical ribbon, to ensure representation of cortical layers, in similar anatomical regions for each case. ROIs were analysed using Fiji software (version 1.51) to obtain a protein load defined as percentage of immunostained area, as in previous studies [30 (link), 37 (link)]. The immunostained area predominantly represents the amount of Aβ accumulated in the brain parenchyma as plaques, and is therefore referred to as such, although CAA might have made a small contribution to this value.