Slide preparation for single-cell-resolution imaging of fluorescent proteins in their three-dimensional near-native environment.

Hugo J. Snippert, Arnout G. Schepers, Gabriele Delconte, Peter D. Siersema, Hans Clevers

Research output: Contribution to journalArticlepeer-review

Abstract

In recent years, many mouse models have been developed to mark and trace the fate of adult cell populations using fluorescent proteins. High-resolution visualization of such fluorescent markers in their physiological setting is thus an important aspect of adult stem cell research. Here we describe a protocol to produce sections (150-200 μm) of near-native tissue with optimal tissue and cellular morphology by avoiding artifacts inherent in standard freezing or embedding procedures. The activity of genetically expressed fluorescent proteins is maintained, thereby enabling high-resolution three-dimensional (3D) reconstructions of fluorescent structures in virtually all types of tissues. The procedure allows immunofluorescence labeling of proteins to depths up to 50 μm, as well as a chemical 'Click-iT' reaction to detect DNA-intercalating analogs such as ethynyl deoxyuridine (EdU). Generation of near-native sections ready for imaging analysis takes approximately 2-3 h. Postsectioning processes, such as antibody labeling or EdU detection, take up to 10 h.

Original languageEnglish
Pages (from-to)1221-1228
Number of pages8
JournalNature Protocols
Volume6
Issue number8
Publication statusPublished - Aug 2011

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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