Figure 3

Digital bioimage analysis workflow for the analysis of epithelial junction proteins and CD4⁺ cells. (a–e) Representative raw and digitalized images of the epithelial junctional proteins (EJPs): (a) E-cadherin, (b) occludin, (c) desmocollin-2, (d) claudin-1, and (e) zonula occludens-1 (ZO-1). Identified EJPs are illustrated in black in the digitalized images. Epithelial tissue was identified based on probability maps generated in Ilastik. EJPs within the epithelial regions were identified in CellProfiler, and the epithelial coverage and mean fluorescence intensity (MFI) were calculated. (f) Representative raw and digitalized images of CD4 expression. CD4⁺ cells were quantified in CellProfiler and classified as either lamina propria (LP) or intra-epithelial (intra-EP) based on the probability maps generated in Ilastik and are illustrated in green in the digitalized images. The first column shows the raw images of each staining, the second column shows the digitalized images of each marker, the third column represents a magnified region in the raw image highlighted in the previous columns, and the fourth column shows the digitalized image of the magnified region. Scale bars represent 100 µm in the first column and 50 µm in the third column.