title: Slide preparation for single-cell-resolution imaging of fluorescent proteins in their three-dimensional near-native environment.
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.
authors.affiliation: Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Hugo J
authors.initials: HJ
authors.lastname: Snippert
authors.forename: Arnout G
authors.initials: AG
authors.lastname: Schepers
authors.forename: Gabriele
authors.initials: G
authors.lastname: Delconte
authors.forename: Peter D
authors.initials: PD
authors.lastname: Siersema
authors.forename: Hans
authors.initials: H
authors.lastname: Clevers
mesh_terms: D007091:Image Processing, Computer-Assisted; D021621:Imaging, Three-Dimensional; D008164:Luminescent Proteins / Q000032:analysis*; D008856:Microscopy, Fluorescence / Q000379:methods; D008867:Microtomy; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D008164:Luminescent Proteins