Superfrost microscope slides

Tissues from parietal cortex from three Alzheimer’s disease patients and three control subjects were obtained from The Netherlands Brain Bank (Amsterdam, The Netherlands) and from the Policlinico Tor Vergata (Rome, Italy), respectively. All patients were clinically diagnosed for absence or presence of Alzheimer’s disease (Supplementary Table 3). For immunohistochemistry, frozen samples were fixed in 4% paraformaldehyde and stored in 20% sucrose plus 0.05% sodium azide until use. Samples were then sectioned at 20 µm, collected on SuperFrost® microscope slides (Sigma-Aldrich) and processed for immunohistochemistry as described below.

5 µm lung and heart sections were cut, from paraffin blocks and mounted on Superfrost® microscope slides (Sigma-Aldrich, Irvine, UK).
Vascular thickening was determined by smooth muscle actin antibody (ab5694, Abcam, Cambridge, UK) staining, thickening was characterised by an increase in the vessel wall diameter of more than 50% of the arterial wall or complete occlusion. The number of remodelled vessels over the total number of vessels present in a lung section was determined. Sections were analysed in a blinded manner.
Heart sections were stained using a common regressive Haemotoxylin and Eosin (H&E) staining methodology to determine gross morphology and cardiomyocyte cross-sectional area. High-power images of five areas randomly distributed across ventricles were analysed by ImageJ software. Mean cardiomyocyte size was determined by measuring the cross sectional area of minimally 20 transversally cut cardiomyocytes at the level of the nucleus.
Picro-sirius red staining was used for analysis of cardiac fibrosis using a Picro-sirius red staining kit (Abcam, Cambridge, UK) as per manufacturer's instructions. High-power images were acquired of five random areas per ventricle. Using ImageJ IHC tool, the percentage tissue area positive for collagen (pink) was determined.