title: Visualization of gene expression in whole mouse retina by in situ hybridization.
abstract: The mouse retinal vasculature provides a powerful model system for studying development and pathologies of the vasculature. Because it forms as a two-dimensional flat plexus, it is easily imaged in its entirety in whole-mount retinal preparations. In order to study molecular signaling mechanisms, it is useful to visualize the expression of specific genes in the entire vascular plexus and retina. However, in situ hybridization on whole-mount retinal preparations is problematic because isolated retinas have a tendency to curl up during hybridization and are difficult to stain. Here we provide a detailed protocol that overcomes these difficulties and visualizes the mRNA distribution of one or two genes in the context of the counterstained retinal vasculature. The protocol takes 3-4 d for single-probe stains, with an additional 2 d for immunohistochemistry co-labeling. In situ hybridization with two probes adds a further 3 d.
authors.affiliation: UCL Institute of Ophthalmology, University College London, London, UK.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Powner
authors.forename: Kristis
authors.initials: K
authors.lastname: Vevis
authors.forename: Jenny A G
authors.initials: JA
authors.lastname: McKenzie
authors.forename: Pranita
authors.initials: P
authors.lastname: Gandhi
authors.forename: Shalini
authors.initials: S
authors.lastname: Jadeja
authors.forename: Marcus
authors.initials: M
authors.lastname: Fruttiger
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002135:Calcium-Binding Proteins; D015870:Gene Expression; D017403:In Situ Hybridization / Q000379:methods*; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D012333:RNA, Messenger / Q000032:analysis; D012160:Retina / Q000502:physiology*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; C519665:DLL4 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D012333:RNA, Messenger