Mcm2 is expressed ubiquitously along the crypt–villus axis and declines slowly as cells differentiate. (A) Sections of normal human (top) and mouse (bottom) small intestine were stained with phalloidin (green) and an antibody against Mcm2 (red). Bars, 200 µm. (B) Mean Mcm2 intensities for segmented nuclei were plotted along the crypt–villus axis for human (left) and mouse (right) tissues. Locations of the crypt and villus domains are indicated. (C) An intestinal crypt stained with Hoechst (blue), phalloidin (green), and an antibody against Mcm2 (red). Individual cells in interphase and mitosis (metaphase and cytokinesis) are outlined by dashed white lines. Bars: (left) 50 µm; (middle and right) 10 µm. (D) Maximum-intensity projections of whole-mount intestinal tissue revealing intestinal crypts and villi (left; Bars, 200 µm). Individual X–Y sections are also shown to reveal the epithelium (right; Bars: [top] 50 µm; [bottom] 100 µm). Tissue was stained with phalloidin (green), Hoechst (blue), and an antibody against Mcm2 (red). The alternating pattern of Mcm2⁺ (green stars) and Mcm2⁻ (orange stars) in the crypt base is highlighted. (E) Images of Lgr5–GFP stem cells (green; top) costained with an Mcm2 antibody (red). Bars, 10 µm. The correlation (Pearson’s correlation R = 0.81, P < 0.0001) between mean Mcm2 and Lgr5–GFP intensities for Lgr5–GFP⁺ cells (n = 69), normalized to the maximum intensity for an individual crypt, is shown. (F) Images of UEA⁺ Paneth cells (top) costained with an Mcm2 antibody (red) and UEA (green). Bars, 10 µm. Mean Mcm2 intensity for segmented nuclei of UEA⁺ Paneth cells was compared with interphase cells (right). (G) Mcm2 (green) and UEA (red) expression in subsets of UEA⁺ cells in crypt and villus domains. Bars, 10 µm. UEA⁺ cells at the crypt base represent Paneth cells. (H) Quantification of mean Mcm2 intensity in individual UEA⁺ cell populations. UEA⁺ cells in the crypt base (Paneth cells, n = 224), in the upper crypt compartment (crypt, n = 132) and in the villus compartment (villus, n = 225) were identified manually, and the nuclear Mcm2 intensity was determined for individual cells (all cells, n = 33,736). There was a significant difference between UEA⁺ cells in the crypt and villus compartments (t test,****, P < 0.0001).