Sandon finesse 325

For paraffin embedding, samples were fixed in 4% PFA, washed in PBS, moved into cassettes (Histosette I, Simport), processed in a Thermo Excelsior AS, and embedded in a Thermo HistoStar machine. A Sandon Finesse 325 (Thermo) microtome was used to cut 5 µm sections which were floated onto Menzel-Glaser SuperFrost Plus slides (Thermo). Slides were stored at 37 o C overnight to dry completely before staining. Hematoxylin and eosin (H&E) and van Gieson's (EVG) stains were performed using a Shandon Varistain 24-4 (Thermo). Slides were removed from machine and mounted with DPX (distyrene, a plasticizer, in toluene-xylene; Sigma) with 24 x 50 mm cover glass (VWR). Slides were imaged on a Zeiss Axio Observer.Z1 with Zen Blue software (Zeiss).
Von Kossa staining was carried out with a Silver plating kit (In Vitro Diagnostic Medical Device) for detection of microcalcification.

For paraffin embedding, samples were fixed in 4% PFA, washed in PBS, moved in to cassettes (Histosette I; Simport), processed in a Thermo Excelsior AS, and embedded in a Thermo HistoStar machine. A Sandon Finesse 325 (Thermo) microtome was used to cut 5-mm sections, which were floated onto Menzel-Glaser Superfrost Plus slides (Thermo). Slides were stored at 37°C overnight to dry completely before staining. Staining with hematoxylin and eosin (H&E) and van Gieson's (EVG) were performed using a Shandon Varistain 24-4 (Thermo). Slides were removed from machine and mounted with DPX (distyrene, a plasticizer, in toluene-xylene; Sigma- Aldrich) with 24 • 50 mm cover glass (VWR). Slides were imaged on a Zeiss Axio Observer.Z1 with Zen Blue software (Zeiss).