4.2. Filipin Staining

Fibroblasts from NPC patients and control individuals were seeded on gelatine-precoated coverslips, incubated for two days at 37 °C and washed thereafter twice with ice-cold PBS. The steps that followed were all performed at room temperature. Here, the cells were fixed with 3% paraformaldehyde (PFA) for 20 min, followed by quenching with 50 mM NH₄Cl for 30 min and finally permeabilised with 0.2% TritonX-100 for further 30 min. Treatment with the cholesterol-binding reagent Filipin (Sigma Aldrich, subsidiary of Merck KGaA St. Louis, Missouri USA) was performed for 2 h in the dark. The images were visualised using a fluorescence microscope (Leica DM IRB).