title: Morphological and immunohistochemical analyses of soluble proteins in mucous membranes of living mouse intestines by cryotechniques.
abstract: We have performed immunohistochemical or ultrastructural analyses of living mouse small intestines using Epon blocks prepared by 'in vivo cryotechnique' (IVCT). By electron microscopy, intracellular ultrastructures of epithelial cells were well preserved in tissue areas 5-10 μm away from cryogen-contact surface tissues. Their microvilli contained dynamically waving actin filaments, and highly electron-dense organelles, such as mitochondria, were seen under the widely organized terminal web. By quick-freezing of fresh resected tissues (FT-QF), many erythrocytes were congested within blood vessels due to loss of blood pressure. By immersion-fixation (IM-DH) and perfusion-fixation (PF-DH), small vacuoles were often seen in the cytoplasm of epithelial cells, and their intercellular spaces were also dilated. Moreover, actin filament bundles were irregular in cross sections of microvilli, compared with those with IVCT. Epon-embedded thick sections were treated with sodium ethoxide, followed by antigen retrieval and immunostained for immunoglobulin A (IgA), Ig kappa light chain (Igκ), J-chain and albumin. By cryotechniques, IgA immunoreactivity was detected as tiny dot-like patterns in cytoplasm of some epithelial cells. Both J-chain and Igκ immunoreactivities were detected in the same local areas as those of IgA. By FT-QF, however, the IgA immunoreactivity was more weakly detected, compared with that with IVCT. In thick sections prepared by IM-DH and PF-DH, it was rarely observed in both plasma and epithelial cells. Another albumin was diffusely immunolocalized in extracellular matrices of mucous membranes and also within blood vessels. Thus, IVCT was useful for preservation of soluble proteins and ultrastructural analyses of dynamically changing epithelial cells of living mouse small intestines.
authors.affiliation: Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan Present address: Department of Occupational Therapy, Health Science University, 7187 Kodachi, Fujikawaguchiko, Yamanashi 401-0380, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Shimo
authors.affiliation: Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan.
authors.forename: Sei
authors.initials: S
authors.lastname: Saitoh
authors.affiliation: Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan.
authors.forename: Yurika
authors.initials: Y
authors.lastname: Saitoh
authors.affiliation: Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan.
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Ohno
authors.affiliation: Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan sohno@yamanashi.ac.jp.
authors.forename: Shinichi
authors.initials: S
authors.lastname: Ohno
mesh_terms: D008841:Actin Cytoskeleton / Q000502:physiology; D000418:Albumins / Q000502:physiology; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis; D001808:Blood Vessels / Q000502:physiology; D015925:Cryopreservation / Q000379:methods*; D004847:Epithelial Cells / Q000502:physiology; D004912:Erythrocytes / Q000502:physiology; D000431:Ethanol / Q000031:analogs & derivatives / Q000494:pharmacology; D007070:Immunoglobulin A / Q000276:immunology / Q000502:physiology; D007144:Immunoglobulin J-Chains / Q000276:immunology / Q000502:physiology; D007145:Immunoglobulin kappa-Chains / Q000276:immunology / Q000502:physiology; D007150:Immunohistochemistry / Q000379:methods*; D007413:Intestinal Mucosa / Q000166:cytology / Q000648:ultrastructure*; D007421:Intestine, Small / Q000166:cytology / Q000648:ultrastructure*; D051379:Mice; D008810:Mice, Inbred C57BL; D008854:Microscopy, Electron / Q000379:methods*; D008871:Microvilli / Q000502:physiology / Q000648:ultrastructure; D008928:Mitochondria / Q000502:physiology / Q000648:ultrastructure; D013194:Staining and Labeling / Q000379:methods; D016707:Tissue Fixation; D014021:Tissue Preservation / Q000379:methods
chemical_list: D000418:Albumins; D001426:Bacterial Proteins; D007070:Immunoglobulin A; D007144:Immunoglobulin J-Chains; D007145:Immunoglobulin kappa-Chains; C098088:sodium ethoxide; D000431:Ethanol
keywords: immunoglobulin A; immunohistochemistry; in vivo cryotechnique; small intestine; ultrastructure