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After incubation with rat anti-tubulin antibody (Cat# ab6160, Abcam, United Kingdom) diluted in 1% bovine serum albumin (BSA) in PBS overnight at 4C, the dishes were washed with PBS for three times and then stained with Alexa Fluor 488 fluorescent secondary antibody (Cat# A32731, Invitrogen, United States) at 37C for 1 h in the dark