For immunocytochemistry, formalin-fixed cells were rinsed with 0.05% Triton X-100 in PBS (PBS-1x) followed by incubation with 2% bovine serum albumin (BSA) in PBS-1x (PBS-1x/BSA) for blocking. Afterward, the cells were incubated with the desired primary antibody diluted in PBS-1x/BSA overnight at 4°C. Following two washes in PBS-1x, samples were incubated with secondary antibodies diluted in PBS-1x/BSA for 1 hour at room temperature. The following primary antibodies were used: rabbit anti-mouse MyoD (Invitrogen), rabbit anti-human MyoD (Invitrogen), and rabbit anti-mouse α-SMA (Abcam). 4′,6-Diamidino-2-phenylindole (DAPI) was used as a nuclear counterstain, and Alexa Fluor 488 or 568 served as the secondary antibody (all from Invitrogen). Imaging was performed by epifluorescence microscopy, consisting of a Xenon lamp, an Axio Zoom V16 microscope, and a Hamamatsu Flash 4.0 v3.

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