Detection of esterified cholesterol in murine Bruch's membrane wholemounts with a perfringolysin O-based cholesterol marker.

Purpose: 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 reliably detect small quantities of neutral lipids including esterified cholesterol (EC). In chorioretinal sections and BrM wholemounts, we tested a novel fluorescent cholesterol marker based on the bacterial toxin perfringolysin O (PFO) and compared results with those obtained with the classic cholesterol dye filipin.

Methods: An engineered plasmid containing the specific cholesterol binding domain (D4) of PFO fused to green fluorescent protein (GFP) was expressed in cultured E.