Morphological and immunohistochemical analyses of soluble proteins in mucous membranes of living mouse intestines by cryotechniques

Satoshi Shimo, Sei Saitoh, Yurika Saitoh, Nobuhiko Ohno, Shinichi Ohno

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)

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 immunoreactivitywas detected as tiny dotlike 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, IVCTwas useful for preservation of soluble proteins and ultrastructural analyses of dynamically changing epithelial cells of livingmouse small intestines.

Original languageEnglish
Pages (from-to)189-203
Number of pages15
JournalMicroscopy
Volume64
Issue number3
DOIs
Publication statusPublished - 06-2015
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Instrumentation
  • Radiology Nuclear Medicine and imaging

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