Immunohistochemical detection of soluble immunoglobulins in living mouse small intestines using an in vivo cryotechnique

Some morphological changes are inevitable during immersion- or perfusion-fixation and following alcohol-dehydration for tissue preparations. Common immunostaining techniques for these specimens have some limitations to capture accurate localizations of soluble proteins in cells and tissues. In this study, to examine in situ distributions of immunoglobulins (Igs), small intestinal tissues of living mice were prepared with our " in vivo cryotechnique" (IVCT). Thin sections were first stained with hematoxylin-eosin for morphology, and then some immunostainings were performed on serial sections for IgA, Ig kappa light chain, IgG1 heavy chain (IgG1), and IgM. Living morphological states of small intestinal tissues, including flowing erythrocytes and opening blood vessels, were observed on paraffin sections prepared with IVCT. IgA was immunolocalized in many plasma cells of the lamina mucosa propria, 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. By perfusion-fixation and alcohol-dehydration, however, IgA immunoreactivity was observed in plasma cells, but not in epithelial cells or the lamina mucosa propria. Thus, IVCT was more useful to examine in vivo immunolocalizations of soluble Igs in small intestines.