GST-PFO labels cholesterol deposits in NPC fibroblasts. (A-C) NPC fibroblasts were cultured in DMEM/10% FBS (A, A’) or in DMEM supplemented with delipidated 10% FBS for 72 h (B-C’). Inset in (B’) shows an enlargement of a merged image of an area marked in (B, B’). Colocalization of GST-PFO and filipin staining is in white. Colocalization of the labels is also seen in vesicles marked by arrows in (B, B’). (C, C’) Omitting GST-PFO during labeling of permeabilized NPC cells yields only traces of non-specific staining with secondary antibodies (C). In these conditions, filipin detects cholesterol accumulated in the cells (C’). (D-D’) Fibroblasts from a healthy donor were cultured in DMEM/delipidated 10% FBS. Cells were fixed, permeabilized with 0.05% Triton X-100 and incubated with 5 μg/ml GST-PFO followed by secondary antibodies conjugated with Texas Red (left) or with 25 μg/ml filipin (right) to compare the staining patterns revealed by the two probes. Scale bar, 20 μm.