Retinal detection of unesterified and esterified cholesterol with filipin. Representative images are shown. A and E: Stains (cyan) for unesterified cholesterol (these sections were treated with filipin). B and F: Control stains for completeness of removal of unesterified cholesterol (these sections were extracted with 70% aqueous ethanol and treated then with filipin). C and G: Control stains for background fluorescence (these sections were extracted with ethanol but not treated with filipin). D and H: Stains (cyan) for esterified cholesterol (these sections were extracted with 70% ethanol and then sequentially treated with cholesterol esterase and filipin). B–D and F–H: Panels for esterified cholesterol consist of phase contrast images (left panels) and histochemistry images (right panels). n = 3 (A–H). Scale bars = 50 μm (A–H). GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; IS, photoreceptor inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, photoreceptor outer segment; RPE, retinal pigment epithelium.