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Visualization of gene expression in whole mouse retina by in situ hybridization.

Nature protocols (2012-05-12)
Michael B Powner, Kristis Vevis, Jenny A G McKenzie, Pranita Gandhi, Shalini Jadeja, Marcus Fruttiger
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.

MATERIALS
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Sigma-Aldrich
Phenol solution, BioReagent, Equilibrated with 10 mM Tris HCl, pH 8.0, 1 mM EDTA, for molecular biology