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.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
Snippert HJ,Schepers AG,Delconte G,Siersema PD,Clevers Hdoi
10.1038/nprot.2011.365subject
Has Abstractpub_date
2011-07-28 00:00:00pages
1221-8issue
8eissn
1754-2189issn
1750-2799pii
nprot.2011.365journal_volume
6pub_type
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