Supplementary macro 2 NUCLEI.ijm

Macro to automate the analysis of Diaminobenzidine/Haematoxylin stained immunohistochemsitry images of nuclear target proteins. <div>The macro imports the images for analysis from one images-only folder into an image stack. It runs a minimum Z-projection to identify the smallest area containing all spheroids and allows the user to crop the extra white space. Afterwards the ImageJ Colour Deconvolution plug-in developed by G Landini based on the method of Ruifrok and Johnston is used to separate the Hematoxylin (H) and DAB staining in the images. In the nuclear stain macro the user would threshold solely the nuclei and not the whole spheroid area. Moreover, the code was optimized to detect solely nuclei by employing Despeckle, Median (2 px), Minimum and Maximum (2 px) filters, Watershed segmentation, as well as a minimum nuclear size (defined in line 55). Macro output from the nuclear staining macro also includes the number of nuclei positive for H and DAB. In the analysis step the percentage positive cells for nuclear stains was derived by dividing the number of DAB-positive nuclei by the number of H-positive nuclei. The macro returns a separate folder with the values of the thresholds, the area and number of nuclei stained for H and DAB, and an image sequence of quality control images with green outlines for the recognized nuclei and magenta outlines for the positive nuclei.