Axio scan z1 digital slide scanner

Immunohistochemistry was performed on FFPE sections (4 μm) as described previously [13] using antibodies listed in Supplementary Information. The slides were scanned using ZEISS Axio Scan.Z1 Digital Slide Scanner (Carl Zeiss Microscopy) with a 40× objective. Image data were acquired using ZEN lite software (Carl Zeiss Microscopy) and quantified using Image J (NIH, Bethesda, MD) with the Color Deconvolution plugin [30] (n = 10). Multi-color immunofluorescence staining was performed using FFPE sections (4 μm). Slides were incubated with a combination of the primary antibodies (CD45, CD31, E-cadherin, pan-cytokeratin (CK), and vimentin) and corresponding secondary antibodies. Slides were quenched with TrueVIEW Autofluorescence Quenching Kit (Vector Laboratories), counterstained with DAPI, and mounted with Vectashield Plus Antifade Mounting Medium (Vector Laboratories). Fluorescently stained slides were scanned using ZEISS Axio Scan.Z1 Digital Slide Scanner (Carl Zeiss Microscopy) with 40× objective. Image data were acquired using ZEN lite software (Carl Zeiss Microscopy). For quantification of E-cadherin + (green)/CK + (red) cells and vimentin + (red)/CK + (green) cells, the number of yellow pixels (green and red overlapping pixels) was counted and normalized with the number of blue pixels (nucleus) per field of view at a final magnification of 40× (n = 4), using Image J.