The melanoma cells were fixed with 2% paraformaldehyde and 0.5% glutaraldehyde (A375 and G361) at 4 °C for 12 h and 0.5% osmic acid for 1 h, and then sequentially dehydrated with 30%, 50%, 70%, 80%, 90%, 100% ethanol and 100% acetone for 10 min. The cells were then treated with 100% propylene oxide for 3 times, 10 min/each time, followed by infiltration with the solution of propylene oxide and epon812 for 1 h, and then epon 812 for 12 h. The sample was transferred to the embedding plate and an appropriate amount of epon 812 was added. The embedded samples were incubated at 60 °C for 48 h in an oven and then proceed for ultrathin section. The sections were first treated 1% H2O2 for 3 times, 10 min/each time, and then blocked with 1% BSA for 15~20 min. Next the sample was incubated with primary antibody at room temperature for 2 h, followed by PBS wash for 3 times, and then blocked with 1% BSA for additional 15~20 min. The sample was then incubated with secondary antibody (1:40 dilution) for 2 h, followed by PBS wash for 10 times, and then rinsed with distilled water for 10 s. After these procedures, the sample was dyed with uranium, and proceeded to TEM imaging.

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