Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, Copenhagen N, Denmark.
Anat Rec (Hoboken). 2011 Sep;294(9):1557-65. doi: 10.1002/ar.21444. Epub 2011 Aug 1.
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.
定量分析肠细胞存在一些挑战，原因如下：细胞密度在整个肠道中存在差异，且可能与年龄有关。一些细胞类型呈分支状，或可改变形状和大小。此外，还需要免疫标记来正确识别细胞类型。免疫标记取决于待标记抗原的上调和下调，以及一抗和二抗、固定和增强程序。在这里，我们提供了一种详细描述的方法，用于对肠道外膜中的 CD169(+)细胞和主要组织相容性复合体 II 抗原 (MHCII(+) )细胞进行免疫标记，并使用基于设计的体视学法对这些细胞进行后续定量分析。我们使用了年轻（5 周龄）和成年（10 周龄）小鼠。与结肠相比，空肠-回肠中的细胞密度更高。在成年动物中，空肠/回肠中细胞密度沿口-肛方向增加，而在幼年动物中，细胞密度在中部最高。在结肠中，两组动物的细胞密度均沿口-肛方向减少。除了结肠中 MHCII(+)细胞的密度外，幼年动物的细胞密度最高。CD169(+)和 MHCII(+)细胞的密度没有差异，除了在幼年动物的结肠中，CD169(+)细胞的密度几乎是 MHCII(+)细胞的两倍。CD169 和 MHCII 抗原似乎在空肠/回肠中由同一细胞同时表达。我们得出结论，细胞密度既取决于小鼠的年龄，也取决于肠道的位置。
