Localizing protein in 3D neural stem cell culture: A hybrid visualization methodology

FieldValue
dc.contributor.authorBennett, Steffany A.L.
dc.contributor.authorFai, Stephen
dc.contributor.authorValenzuela, Nicolas
dc.contributor.authorImbeault, Sophie
dc.date.accessioned2011-01-18T14:42:21Z
dc.date.available2011-01-18T14:42:21Z
dc.date.created2010
dc.date.issued2010
dc.identifier.urihttp://hdl.handle.net/10393/19699
dc.identifier.urihttp://www.jove.com/index/Details.stp?ID=2483
dc.description.abstractThe importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro.
dc.language.isoen
dc.subjectneuroscience
dc.subjectneural stem cell
dc.subjecthippocampus
dc.subjectcryosectioning
dc.subject3D modelling
dc.subjectneurosphere
dc.subjectMaya
dc.subjectcompositing
dc.titleLocalizing protein in 3D neural stem cell culture: A hybrid visualization methodology
dc.typeArticle
dc.identifier.doi10.3791/2483
CollectionBiochimie, microbiologie et immunologie // Biochemistry, Microbiology and Immunology
Publications en libre accès financées par uOttawa // uOttawa financed open access publications

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