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Using methanol to preserve retinas for immunostaining

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Using methanol to preserve retinas for immunostaining

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Abstract

Background Experimental studies on retinal vasculature and retinal ganglion cells (RGCs) have investigated developmental and pathological conditions of the retina; they rely on whole-mount retinal immunostaining. Methanol, an auxiliary fixed medium for retinal whole-mount preparations, has been used in some studies; however, its application in short- and long-term storage of retinas for further study has not been well described. We aimed to evaluate methanol use as a preservation treatment for further immunostaining of the retina. Methods We generated the oxygen-induced retinopathy (OIR) and optic nerve crush (ONC) mouse models and used their retinas for analysis. We pipetted cold methanol (−20 °C) on the surface of the retina to help fix the tissues while promoting permeability, after which the retinas were stored in cold methanol (−20 °C) for 1, 6, or 12 months before being evaluated using various optical techniques. Thereafter, retinal whole-mount immunostaining was performed to analyse retinal neovascularisation and retinal hypoxia in OIR model, and retinal ganglion cell survival rate in ONC model. Results Quantitative analysis revealed no significant differences in these fixed retinas after long-term storage in terms of retinal vasculature or retinal hypoxia in OIR model. Similarly, no significant difference was found in RGC survival rate after long-term storage in methanol. These results suggest that methanol can be used as a storage medium when preserving retinal whole-mount samples. Conclusions Cold (−20 °C) methanol is a good medium for storing fixed retinas for long periods, which is useful when retinas must be stored for further analysis.

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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.
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In ischemic retinopathies, unrelieved hypoxia induces the formation of architecturally abnormal, leaky blood vessels that damage retina and ultimately can cause blindness. Because these newly formed blood vessels are functionally defective, they fail to alleviate underlying hypoxia, resulting in more pathological neovascularization and more damage to retina. With an established model of ischemic retinopathy, we investigated inhibition of glycogen synthase kinase-3β (GSK-3β) as a means for improving the architecture and functionality of pathological blood vessels in retina. In vitro, hypoxia increased GSK-3β activity in retinal endothelial cells, reduced β-catenin, and correspondingly impaired integrity of cell/cell junctions. Conversely, GSK-3β inhibitors restored β-catenin, improved cell/cell junctions, and enhanced the formation of capillary cords in three-dimensional collagen matrix. In vivo, GSK-3β inhibitors, at appropriately moderate doses, strongly reduced abnormal vascular tufts, reduced abnormal vascular leakage, and improved vascular coverage and perfusion during the proliferative phase of ischemia-driven retinal neovascularization. Most importantly, these improvements in neovasculature were accompanied by marked reduction in retinal hypoxia, relative to controls. Thus, GSK-3β inhibitors offer a promising strategy for alleviating retinal hypoxia by correcting key vascular defects typically associated with ischemia-driven neovascularization.
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The listing of cell types present in the retina is nearing completion, the first time this can be said for any significantly complex sample of the central nervous system. Mammalian retinas contain approximately 55 separate neuronal types. The functions of 22 of them are known or can be strongly inferred. For these 22, in every instance, a cell defined as a 'type' by structural criteria carries out a distinct and individual physiological function. Electrophysiological experiments continue to reveal new features of the retina's handling of information, and there is every reason to believe that the remaining 33 types of cell will also have distinct physiological functions. Further subtleties clearly exist in both peripheral and central visual coding.
The RNA binding protein RBPMS is a selective marker of ganglion cells in the mammalian retina
  • AR Rodriguez
  • LP Sevilla Muller
  • NC Brecha