Counting rhodopsin-positive phagosomes residing in the retinal pigment epithelium (RPE) in the eye at different times of day allows a quantitative assessment of engulfment and digestion phases of diurnal RPE phagocytosis, which efficiently clears shed photoreceptor outer segment fragments (POS) from the neural retina. Comparing such activities among age- and background-matched experimental wild-type and mutant mice or rats serves to identify roles for specific proteins in the phagocytic process. Here, we describe experimental procedures for mouse eye harvest, embedding, sectioning, immunofluorescence labeling of rod POS phagosomes in RPE cells in sagittal eye sections, imaging of POS phagosomes in the RPE by laser scanning confocal microscopy, and POS quantification.
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This work was supported by NIH grant EY013295.
Mullen RJ, LaVail MM (1976) Inherited retinal dystrophy: primary defect in pigment epithelium determined with experimental rat chimeras. Science 192:799–801PubMedCrossRefGoogle Scholar
Nandrot EF, Kim Y, Brodie SE, Huang X, Sheppard D, Finnemann SC (2004) Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking αvβ5 integrin. J Exp Med 200:1539–1545PubMedCrossRefGoogle Scholar
LaVail MM (1976) Rod outer segment disk shedding in rat retina: relationship to cyclic lighting. Science 194:1071–1074PubMedCrossRefGoogle Scholar
Young RW, Bok D (1969) Participation of the retinal pigment epithelium in the rod outer segment renewal process. J Cell Biol 42:392–403PubMedCrossRefGoogle Scholar
Bosch E, Horwitz J, Bok D (1993) Phagocytosis of outer segments by retinal pigment epithelium: phagosome- lysosome interaction. J Histochem Cytochem 41:253–263PubMedCrossRefGoogle Scholar
Gibbs D, Kitamoto J, Williams DS (2003) Abnormal phagocytosis by retinal pigmented epithelium that lacks myosin VIIa, the Usher syndrome 1B protein. Proc Natl Acad Sci U S A 100:6481–6486PubMedCrossRefGoogle Scholar
Nandrot EF, Anand M, Almeida D, Atabai K, Sheppard D, Finnemann SC (2007) Essential role for MFG-E8 as ligand for αvβ5 integrin in diurnal retinal phagocytosis. Proc Natl Acad Sci U S A 104:12005–12010PubMedCrossRefGoogle Scholar
Adamus G, Zam ZS, Arendt A, Palczewski K, McDowell JH, Hargrave PA (1991) Anti-rhodopsin monoclonal antibodies of defined specificity: characterization and application. Vision Res 31:17–31PubMedCrossRefGoogle Scholar