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M. C. Dettbarn, J. Knobloch, S. Grisanti, M. Rudolf; Detection of Esterified Cholesterol in Murine Bruch’s Membrane Wholemounts with a Novel Cholesterol Dye. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4937.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the effects of Bruch’s membrane (BrM) neutral lipid deposition in mouse models and its significance to aging and age-related macular degeneration it is essential to reliable detect lipids including esterified cholesterol (EC). In murine BrM wholemounts we tested the novel fluorescent cholesterol dye GFP-D4 derived from the bacterial toxin Perfringolysin O (PFO) and compared results to those obtained with filipin.
An engineered plasmid containing the specific cholesterol binding domain (D4) of PFO fused to green fluorescent protein (GFP) was expressed in cultured E. coli. GFP-D4 was isolated, purified, and concentrated. Ten BrM-choroid wholemounts of 8-10-month-old ApoEnull mice were 1) prepared with clean removal of RPE and retina, 2) stained with GFP-D4 (80 µg/ml) for EC following ethanol extraction and cholesterol esterase treatment and 3) examined by epifluorescence microscopy.
The fluorescence intensity of freshly isolated GFP-D4 was strong, stable, and superior to filipin. In all specimens we could sharply locate the GFP-D4 signal to BrM. Across a wholemount sample we found a laminar pattern of multiple defined dots representing EC in lipid vesicles present in aged murine BrM. A semi-quantitative evaluation of BrM lipid deposition is possible by measuring GFP-D4 fluorescence intensity.
GFP-D4 allowed a robust and direct detection of EC in aged murine BrM. In wholemount samples its strong and stable fluorescence facilitated a semi-quantitative evaluation of BrM’s EC content over a large area. The patterns of EC deposition in murine BrM wholemounts are comparable to findings in human BrM wholemounts (Rudolf et al. 2009). GFP-D4 could be an important tool for investigating the effects of BrM lipid deposition in mouse models.
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