Pannoramic flash 3 system

Fixed lung, liver, and tumor tissues were paraffin-embedded and cut into 4 μm thick sections. Histological processing of specimens was carried out by dewaxing, staining with H&E, rehydrating, and sealing to attach to glass slides. Anti-mitochondria IHC staining to visualize metastasis was performed according to previously published protocols.^{12 (link)} Briefly, lung and liver slides were stained with the anti-human mitochondria antibody (AbCAM, Cambridge, MA, Cat# ab92824) with a 1:1000 dilution overnight after blocking with 10% of horse serum. The following day, the slides were incubated with the secondary anti-mouse antibody, visualized with the DAB agent (Sigma-Aldrich, Cat# D5637), counterstained in Gill’s hematoxylin, and mounted with Permount mounting media. Representative tissue images were captured using a Keyence BZ-X700 microscope. Representative whole lung or liver images were digitally scanned using a Pannoramic FLASH III system (3D Histech). Lung or liver metastatic burden of each mouse was quantified by measuring the percentage of the metastasis area in 3–4 representative fields per H&E staining tissue with the Keyence Hybrid Cell Count module.

Fixed tumors and organs were embedded in paraffin and unstained slides were prepared for immunostaining or stained with H&E. IHC staining was performed as previously described (21 (link)). Primary antibodies included rabbit anti-Ki67 (1:400), rabbit anti-CD31 (1:100) (#9027; #77699, Cell Signaling Technology), rabbit anti-cleaved PARP (1:50) and rabbit anti-cleaved-caspase-3 (1:200), detected by a biotinylated horse anti-rabbit IgG antibody (BA-1100, Vector Laboratories Inc., Burlingame, CA) secondary antibody. Anti-human-specific mitochondria IHC staining (AbCAM, Cambridge, MA, cat# ab92824) was performed at a 1:1000 dilution to visualize metastases. All slides were developed with DAB Impact (Vector Labs, Burlingame, CA), counterstained in Gill’s hematoxylin (Vector Labs) and mounted with Cytoseal XYL mounting media. Tissue images were acquired with a Keyence BZ-X700 microscope. Quantification of metastatic burden was performed by digital scanning of whole stained slides using a Pannoramic FLASH III system (3D Histech), followed by manual counting of metastatic lesions present in the tissue section. Quantification of Ki67-, CD31-, cleaved-PARP- and cleaved-caspase-3-positive tumor cells was performed by calculating the area of positive cells in 4–5 representative fields per section using the Keyence Hybrid Cell Count module.