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. [1 ]
Schepers, Arnout G. [1 ]
Delconte, Gabriele [2 ]
Siersema, Peter D. [2 ]
Clevers, Hans [1 ]
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.
引用
收藏
页码:1221 / 1228
页数:8
相关论文
共 26 条
[1]   Identification of stem cells in small intestine and colon by marker gene Lgr5 [J].
Barker, Nick ;
van Es, Johan H. ;
Kuipers, Jeroen ;
Kujala, Pekka ;
van den Born, Maaike ;
Cozijnsen, Miranda ;
Haegebarth, Andrea ;
Korving, Jeroen ;
Begthel, Harry ;
Peters, Peter J. ;
Clevers, Hans .
NATURE, 2007, 449 (7165) :1003-U1
[2]   Lgr5+ve Stem Cells Drive Self-Renewal in the Stomach and Build Long-Lived Gastric Units In Vitro [J].
Barker, Nick ;
Huch, Meritxell ;
Kujala, Pekka ;
van de Wetering, Marc ;
Snippert, Hugo J. ;
van Es, Johan H. ;
Sato, Toshiro ;
Stange, Daniel E. ;
Begthel, Harry ;
van den Born, Maaike ;
Danenberg, Esther ;
van den Brink, Stieneke ;
Korving, Jeroen ;
Abo, Arie ;
Peters, Peter J. ;
Wright, Nick ;
Poulsom, Richard ;
Clevers, Hans .
CELL STEM CELL, 2010, 6 (01) :25-36
[3]   GREEN FLUORESCENT PROTEIN AS A MARKER FOR GENE-EXPRESSION [J].
CHALFIE, M ;
TU, Y ;
EUSKIRCHEN, G ;
WARD, WW ;
PRASHER, DC .
SCIENCE, 1994, 263 (5148) :802-805
[4]   The IRG mouse: A two-color fluorescent reporter for assessing Cre-mediated recombination and imaging complex cellular relationships in situ [J].
De Gasperi, Rita ;
Rocher, Anne B. ;
Sosa, Miguel A. Gama ;
Wearne, Susan L. ;
Perez, Gissel M. ;
Friedrich, Victor L., Jr. ;
Hof, Patrick R. ;
Elder, Gregory A. .
GENESIS, 2008, 46 (06) :308-317
[5]   Artifacts in frozen section preparation [J].
Desciak, EB ;
Maloney, ME .
DERMATOLOGIC SURGERY, 2000, 26 (05) :500-504
[6]   A Cre-Reporter Transgenic Mouse Expressing the Far-Red Fluorescent Protein Katushka [J].
Dieguez-Hurtado, Rodrigo ;
Martin, Javier ;
Martinez-Corral, Ines ;
Dolores Martinez, Maria ;
Megias, Diego ;
Olmeda, David ;
Ortega, Sagrario .
GENESIS, 2011, 49 (01) :36-45
[7]   Technicolour transgenics: Imaging tools for functional genomics in the mouse [J].
Hadjantonakis, AK ;
Dickinson, ME ;
Fraser, SE ;
Papaioannou, VE .
NATURE REVIEWS GENETICS, 2003, 4 (08) :613-625
[8]   Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system [J].
Livet, Jean ;
Weissman, Tamily A. ;
Kang, Hyuno ;
Draft, Ryan W. ;
Lu, Ju ;
Bennis, Robyn A. ;
Sanes, Joshua R. ;
Lichtman, Jeff W. .
NATURE, 2007, 450 (7166) :56-+
[9]   Faithful activation of an extra-bright red fluorescent protein in "knock-in" Cre-reporter mice ideally suited for lineage tracing studies [J].
Luche, Herve ;
Weber, Odile ;
Rao, Tata Nageswara ;
Blum, Carmen ;
Fehling, Hans Joerg .
EUROPEAN JOURNAL OF IMMUNOLOGY, 2007, 37 (01) :43-53
[10]   Activation of EGFP expression by Cre-mediated excision in a new ROSA26 reporter mouse strain [J].
Mao, XH ;
Fujiwara, Y ;
Chapdelaine, A ;
Yang, HD ;
Orkin, SH .
BLOOD, 2001, 97 (01) :324-326