Imaging and Quantification Techniques for Biological Samples

Digital images were captured on a Nikon TiE inverted microscope (Nikon Instruments), a Zeiss Observer.Z1 inverted microscope (Zeiss) or a Zeiss LSM 710 confocal microscope (Zeiss). The motorized stage and automated image-stitching parameters were used to obtain complete images of each tumour/tissue array. Immunohistochemical stains were captured using brightfield optics and a Nikon DSRi2 colour camera at ×20 magnification. Multi-channel immunofluorescent samples were captured on a CCD Hammamatsu Camera at ×10 magnification and an AxioCam MRm (Zeiss) at ×10 and ×20 magnification, using independently controlled excitation and emission wavelength filter-wheels for each specific fluorophore. NIS Elements software v.4.60.00 (Nikon) and Zeiss Zen 3.0 software (Zeiss) were used for image quantification and pseudo-colouring of immunofluorescent images where appropriate. Also, ImageJ Fiji^{81 (link)} was used for image quantification. Parameters in the tube formation assay were analysed in ImageJ Fiji using a code for the morphometric analysis of ‘Endothelial Tube Formation Assay’^{82 (link)}. Imaris 9.8.1 imaging software was used to analyse images capture by confocal/multiphoton microscope.