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

H.J.G. Snippert, A.G. Schepers, G. Delconte, P.D. Siersema, H. Clevers

Research output: Contribution to journal/periodicalArticleScientificpeer-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 mum) 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 mum, 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. [KEYWORDS: Image Processing, Computer-Assisted, Imaging, Three-Dimensional, Luminescent Proteins/ analysis, Microscopy, Fluorescence/methods, Microtomy, Single-Cell Analysis/ methods]
Original languageEnglish
Pages (from-to)1221-1228
JournalNature Protocols
Volume6
Issue number8
DOIs
Publication statusPublished - 2011

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