Figure 1.

Annotation workflow for definition of regions. After MALDI MSI data acquisition, any residual matrix was removed and the tissue section histologically stained with hematoxylin and eosin. A consecutive section was stained with Masson’s trichrome to detect collagen (in blue). Two additional consecutive tissue sections were used for immunofluorescence of α-SMA and CD68 to detect VSMCs and macrophages, respectively. The histological image was then annotated based on the information gained from Masson’s trichrome and the immunofluorescence images; seven regions were defined: calcification (purple), hemorrhage (brown), lipid-necrotic core (black), collagen (yellow), macrophages (orange), outer VSMCs (cyan) and inner VSMCs (green).