Quantitative Immunohistochemical Analysis

Representative images of the immunohistochemical stainings were obtained using a digital slide scanner (3DHISTECH, Sysmex, Budapest, Hungary). Subsequently, separate image sections of the tumor and tumor-surrounding normal colorectal tissue were created at a 10-fold magnification using the Case Viewer program (3DHISTECH, Sysmex, Budapest, Hungary). The semi-quantitative analysis of the images was carried out using ImageJ (NIH) as previously described [22 (link)]. In brief, the images were converted into 8-bit greyscale images and inverted. Each pixel of the image was assigned an intensity value between 0 and 255. To exclude non-specific low background pixels and intensities from the analysis, a lower threshold of 50 was set. Finally, the mean intensity values for all pixels of an image were generated by measurements in ImageJ.