Before sectioning, tissue blocks were transferred to a freezer at −20°C for 24 hours. Samples were sectioned on a microtome (Thermo Fisher Scientific HM550 Cryostat) at −18° to −20°C. Three serial (or as close to serial as possible) sections were cut from each sample in the following order: two 12-μm-thick sections mounted (Fisherbrand Superfrost/Plus microscope slides) for histological analysis (H&E and von Kossa/Nuclear Fast Red, respectively) and one thicker section (20 μm) mounted on a fused quartz slide (Technical Glass Products) for correlative analyses (Raman microscopy, SE/BSE/EDS). Histological sections were air-dried and baked at 65°C for 45 min to adhere the sample to the slide and were processed at the Cornell College of Veterinary Medicine Animal Health Diagnostic Center using standard protocols for the stains. The thicker section was not baked (to preserve structure) and was stored in a desiccator for no more than a month before and during transport to Raman facilities.
All sections (histology and unstained) were imaged using a ScanScope CS0 (Aperio, Vista, CA) at ×40 magnification. Stained sections were then reviewed carefully to select regions of interest containing calcifications based on von Kossa staining (118 (link)). The histological classifications of calcification regions are described in Supplementary Text (“Calcification classifications” section).
All sections (histology and unstained) were imaged using a ScanScope CS0 (Aperio, Vista, CA) at ×40 magnification. Stained sections were then reviewed carefully to select regions of interest containing calcifications based on von Kossa staining (118 (link)). The histological classifications of calcification regions are described in Supplementary Text (“Calcification classifications” section).