共 26 条
Slide preparation for single-cell-resolution imaging of fluorescent proteins in their three-dimensional near-native environment
被引:26
作者:

Snippert, Hugo J.
论文数: 0 引用数: 0
h-index: 0
机构:
Hubrecht Inst KNAW, Utrecht, Netherlands Hubrecht Inst KNAW, Utrecht, Netherlands

Schepers, Arnout G.
论文数: 0 引用数: 0
h-index: 0
机构:
Hubrecht Inst KNAW, Utrecht, Netherlands Hubrecht Inst KNAW, Utrecht, Netherlands

Delconte, Gabriele
论文数: 0 引用数: 0
h-index: 0
机构:
Univ Med Ctr Utrecht, Dept Gastroenterol & Hepatol, Utrecht, Netherlands Hubrecht Inst KNAW, Utrecht, Netherlands

Siersema, Peter D.
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机构:
Univ Med Ctr Utrecht, Dept Gastroenterol & Hepatol, Utrecht, Netherlands Hubrecht Inst KNAW, Utrecht, Netherlands

论文数: 引用数:
h-index:
机构:
机构:
[1] Hubrecht Inst KNAW, Utrecht, Netherlands
[2] Univ Med Ctr Utrecht, Dept Gastroenterol & Hepatol, Utrecht, Netherlands
关键词:
MARKS STEM-CELLS;
REPORTER MOUSE;
SMALL-INTESTINE;
HAIR FOLLICLE;
EXPRESSION;
CRE;
MICE;
LGR5;
ACTIVATION;
SKIN;
D O I:
10.1038/nprot.2011.365
中图分类号:
Q5 [生物化学];
学科分类号:
071010 ;
081704 ;
摘要:
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 mu m) 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 mu m, 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.
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收藏
页码:1221 / 1228
页数:8
相关论文
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