Epi-fluorescence Imaging of Glycolipid Analogs

For the epi-fluorescence imaging of ATTO594-NeuAcLc₄Cer and ATTO594-Lc₄Cer incorporated in cell plasma membranes (PMs), the fluorescent analogs were first dispersed in HBSS (Nissui), buffered with 2 mM PIPES (Dojindo) at pH 7.4 (P-HBSS) at a final concentration of 1.0 μM, and the dispersed solution was incubated with T24 cells on a glass-base dish (Iwaki) at 22 °C for 15 min. Then, the cells were chilled on ice-water (2.8 °C), washed three times with pre-chilled P-HBSS, and incubated in pre-chilled P-HBSS containing 1% (v/v) Triton X-100 on ice-water for 15 min.^{28 (link)} Then, the cells were washed three times with cold P-HBSS, washed twice with pre-chilled PBS, fixed with 4% paraformaldehyde in PBS for 90 min, and observed under a Nikon Ts-2FL epi-fluorescence microscope equipped with a 60× 1.40 NA oil objective and a high-sensitive CMOS sensor-based camera (Nikon, DS-Qi2).