To evaluate cell retention of the probe post-fixation/permeabilization, LX2 cells (2 × 104) were plated on a 12 mm glass coverslip placed in a well of a 24 well-plate and incubated for 24 h at 37°C in an atmosphere of 5% CO2. The cells were then treated with 500 ηM NRB-AF12 for 30 min, fixed with 4% paraformaldehyde for 15 min at room temperature and immediately mounted on microscope slides, or fixed with 4% paraformaldehyde, permeabilized with either 0.1% Triton X-100 (10 min at room temperature) or ice cold methanol (5 min at −20°C), and subsequently mounted.