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

TY - JOUR

T1 - Slide preparation for single-cell-resolution imaging of fluorescent proteins in their three-dimensional near-native environment

AU - Snippert, H.J.G.

AU - Schepers, A.G.

AU - Delconte, G.

AU - Siersema, P.D.

AU - Clevers, H.

N1 - Reporting year: 2011

PY - 2011

Y1 - 2011

N2 - 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]

AB - 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]

U2 - 10.1038/nprot.2011.365

DO - 10.1038/nprot.2011.365

M3 - Article

SN - 1754-2189

VL - 6

SP - 1221

EP - 1228

JO - Nature Protocols

JF - Nature Protocols

IS - 8

ER -