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.

Slide preparation for single-cell-resolution imaging of fluorescent proteins in their three-dimensional near-native environment / H.J. Snippert, A.G. Schepers, G. Delconte, P.D. Siersema, H. Clevers. - In: NATURE PROTOCOLS. - ISSN 1754-2189. - 6:8(2011 Aug), pp. 1221-1228.

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

G. Delconte;
2011-08

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.
marks stem-cells; reporter mouse; small-intestine; hair follicle; expression; CRE; mice; LGR5; activation; skin
Settore MED/12 - Gastroenterologia
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/330636
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