Standard α-MEM was exchanged with α-MEM supplemented with 100 nM SiR-actin (633 nm, Spirochrome, Switzerland) and 10 μM verapamil (Spirochrome, Switzerland) 24 hours before imaging and then incubated at 37°C in a humidified atmosphere with 5% CO2. Samples were imaged after 5, 10, 15, and 32 days of tissue culture with a Leica SP8 confocal microscope using a 25× 0.95-NA water immersion objective lens (Leica Fluotar VISIR) and a helium-neon laser at 633 nm. After each imaging cycle, medium containing SiR-actin was exchanged with standard α-MEM to reduce prolonged SiR-actin exposure.

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