Abstract
PURPOSE: The subretinal layer between apical retinal pigment epithelium (RPE) and the apices of the photoreceptor outer segments is important to aging and degenerative pathogenesis, but current protocols do not provide intact horizontal images of this retinal space. Thus, an RPE/retina whole mount staining protocol was developed to observe integral subretinal regions. METHODS: RPE/retina whole mounts were stained instead of separated retina or RPE whole mounts. Hydrogen peroxide (H(2)O(2)) treatment was applied in different conditions of concentration, time, and temperature for the bleaching of RPE and choroidal melanocyte pigmentation in the pigmented RPE/retina whole mounts before antibody staining. RESULTS: An RPE/retina whole mount staining protocol provided better morphology of the photoreceptor outer segment than current retina whole mount. For the pigmented eyes, 10% H(2)O(2) pretreatment effectively bleached melanin at 55°C less than 2 hours, or at 4°C within 7 days, without significant effect on immunolabeling efficacy of most antibodies tested. Actually, 55°C bleaching improved immunolabeling intensities compared to the nonbleaching control. This melanin bleaching RPE/retina protocol was applied further to observe macrophage/microglia extending from the sclera to outer plexiform layer in the CX3CR1+/GFP retina. CONCLUSIONS: The pigment bleaching RPE/retina whole mount allowed integral horizontal imaging between choroid to photoreceptor layers, which could not be accomplished with existing methods of separated retina or RPE whole mount. Under these procedures, antigenicity of most antibodies also was well preserved. TRANSLATIONAL RELEVANCE: This efficient protocol provides a tool to observe an integral view of subretinal structures including macrophage/microglia and transplanted cells, and further allows study of the interrelationship between the choroid and photoreceptor in models of aging, disease, and retinal degeneration.