Quantitative assessment of macrophages in the muscularis externa of mouse intestines

Quantification of intestinal cells is challenging for several reasons: The cell densities vary throughout the intestines and may be age dependent. Some cell types are ramified and/or can change shape and size. Additionally, immunolabeling is needed for the correct identification of cell type. Immunolabeling is dependent on both up- and down-regulation of the antigen being labeled as well as on the primary and secondary antibodies, the fixation, and the enhancement procedures. Here, we provide a detailed description of immunolabeling of CD169(+) cells and major histocompatibility class II antigen (MHCII(+) ) cells and the subsequent quantification of these cells using design-based stereology in the intestinal muscularis externa. We used young (5-weeks-old) and adult (10-weeks-old) mice. Cell densities were higher in jejunum-ileum, when compared with colon. In jejunum/ileum, the cell densities increased in oral-anal direction in adults, whereas the densities were highest in the midpart in young animals. In colon, the cell densities decreased in oral-anal direction in both groups of animals. Except for the density of MHCII(+) cells in colon, the cell densities were highest in young animals. Densities of CD169(+) and MHCII(+) cells did not differ, except in the colon of young animals where the CD169(+) density was almost twice as high as the MHCII(+) density. CD169 and MHCII antigens seem to be expressed simultaneously by the same cell in jejunum/ileum. We conclude that cell densities depend on both the age of the mouse and on the location in the intestines.