Immunohistochemical Detection of Soluble Immunoglobulins in Small Intestines

We performed immunohistochemical or ultrastructural analyses of living mouse small intestines prepared by “in vivo cryotechnique” (IVCT). Living morphological states of small intestinal tissues, including flowing erythrocytes and opening blood vessels, were observed on paraffin-embedded sections prepared with IVCT. IgA was immunolocalized in many plasma cells of the lamina propria mucosa, intestinal matrices, and also in epithelial cells of the intestinal villi and crypts. Both IgG1 and IgM immunoreactivities were mainly detected in blood vessels, whereas only IgG1 was also immunolocalized in interstitial matrices of mucous membranes. Confocal laser scanning micrographs of double-fluorescence immunostaining for IgA immunoreactivity are detected in the cytoplasm of epithelial cells as well as plasma cells in the lamina propria mucosa. On the other hand, by electron microscopy, intracellular ultrastructures of epithelial cells were well preserved in tissue areas 5–10 μm away from the cryogen-contact surface tissues. Apical microvilli of epithelial cells contained dynamically waving actin filaments. Furthermore, highly electron-dense organelles, such as mitochondria, in addition to endoplasmic reticulum and ribosomes, were well preserved under the widely organized terminal web. Additionally, Epon-embedded thick sections were treated with sodium ethoxide, followed by antigen retrieval, and immunostained for various proteins, such as IgA, Igκ, IgG1, IgM, J-chain, and albumin. IgA immunoreactivity was detected as a tiny dot-like pattern in the cytoplasm of some epithelial cells and plasma cells localized in the lamina propria. The J-chain and Igκ immunoreactivities were also detected in the same local areas as those of IgA. Thus, IVCT was useful for the preservation of soluble serum proteins and ultrastructural analyses of dynamically changing epithelial cells of living mouse small intestines.