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B. Xie, J.-K. Shen, M. Swaim, P. Campochiaro; In Vivo Retinal Immunostaining. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4058.
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The purpose of this study was to identify ways to improve qualitative and quantitative assessments of retinal vessels and neovascularization.
At P17, mice with oxygen-induced ischemic retinopathy were given an intravitreous injection of one of a variety of florescence-labeled or unlabeled antibodies. For double staining, equal volumes of two antibodies were pre-mixed. Mice were euthanized 8 to 12 hours after injection and eyes were fixed in formalin for 5 hours. Retinas were dissected, washed, and flat mounted (retinas from eyes injected with labeled antibodies) or incubated with secondary antibody at room temperature for 45 minutes and then flat mounted (retinas from eyes injected with unlabeled antibodies).
Retinas from eyes injected with labeled anti-PECAM antibody showed good resolution of the fine structure of retinal neovascularization including filopodia at the tips of sprouts and areas of aneurismal dilation within some stalks. Sprouts were seen from both superficial and deep retinal vessels, which were also stained with this technique. Retinas from eyes injected with unlabeled anti-PECAM antibody and then incubated with labeled secondary antibody showed selective staining of retinal neovascularization with little or no background, greatly facilitating identification and quantification of the neovascularization by image analysis software. Double labeling with anti-PECAM and one of three other antibodies, anti-CD45, F4/80, and anti-CXCR4 showed exquisite localization of various populations of bone marrow-derived cells with respect to the vasculature.
This study describes techniques that facilitate measurements and detailed structural analysis of retinal neovascularization. They also allow identification and quantification of populations of bone-marrow derived cells and study of their relationship to growing and regressing vessels.
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