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JG Hollyfield, KG Shadrach, ME Rayborn; Development of Methods to Isolate Drusen from Bruch’s Membrane . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2786.
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Purpose: Although a few studies using immunocytochemical methods have identified some of the specific molecules present in drusen, in order to define with state of the art biochemical techniques the full complement of molecules contained in drusen, a reliable method for drusen isolation is needed. The purpose of this study was to develop such a drusen isolation procedure. Methods: Frozen normal and age-related macular degeneration donor eyes (41 total) from 60 to 103 years of age were used. When thawed, the anterior segment was removed with a circumferential cut posterior to the limbus. After removing the retina and vitreous, the RPE was gently brushed from the inner surface exposing Bruch's membrane. The choroid was then stripped from the sclera and removed to a plastic petri dish. The area of the Bruch's membrane from within the arcades can be separated with a 6 mm trephine and maintained separately from the peripheral sample. At 20 to 40 diameter magnifications, the surface of Bruch's membrane was scanned for the presence of drusen. Finely sharpened watchmakers forceps and small glass rods are sufficient to dislodge drusen from Bruch's membrane. Drusen can be picked up with 200-400 µm diameter glass pipets for transfer to small centrifuge tubes. Results: Drusen accumulated with this method can yield up to 10 µg of protein from a single eye. Furthermore, drusen of different shape, size and color can be sorted for separate analysis. Microscopic studies of isolated drusen reveal a variety of different forms, including amorphous, membranous or granular content. Sufficient protein is present in drusen isolates for SDS/PAGE, western blotting and/or QTOF mass spectrometric analysis. Conclusion: This microdissection procedure is successful in isolating sufficient drusen for a variety of analytical techniques. The full complement of drusen proteins and lipids should be forthcoming from analysis of different types of drusen isolated with this procedure.
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