Immunohistochemical Analysis of Tissue Samples

For H&E staining, sections were stained with Gill's hematoxylin and eosin Y and then scanned at 20X using a Zeiss AxioScan.Z1 slide scanner. The following primary antibodies were used for IHC/ICC¹⁵: rabbit anti-Ki67 (1:5,000; Abcam); rabbit anti-activated caspase-3 (1:200; R&D Systems); rabbit anti-CD31 (Santa Cruz Biotechnology); mouse anti-skeletal myosin (1:400; Sigma); rabbit anti-β1 (1:200; Abgent); mouse anti-fyn (1:100; BioLegend); mouse anti-CD44 (1:100; AbD Serotec); rabbit anti-E-cadherin (1:200; Cell Signaling Technology); mouse anti-human nuclear antigen (HNA; 1:100; Millipore). Secondary antibodies were Alexa-568-conjugated goat anti mouse/rabbit, unless stated otherwise (1:500; Invitrogen). Samples were mounted in Prolong Gold with DAPI (Invitrogen). Some samples were stained with Alexa-633-phalloidin (1:25; Invitrogen).23 (link) Samples were viewed using 20× objectives on a Nikon Eclipse TE200 fluorescent microscope, or Zeiss Axio Observer.Z1 microscope with LSM 710 confocal laser scanner.