Immunocytochemistry (ICC)

C2C12 cells were cultured on collagen-coated 25 mm coverslips in 6-well Petri plates. At designated time points, the culture media was removed by aspiration and cells were washed three times with 1X PBS. Cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 minutes, then quickly washed twice with 1X PBS. Fixed cells were incubated with a blocking solution of 5% bovine serum albumin (BSA) in 1X PBS for 1 hour, followed by overnight incubation with the primary antibody at 4°C. The next day, cells were washed and incubated with a fluorescently labeled species-specific secondary antibody for the corresponding primary antibody, as indicated in the figure legends. The secondary antibody was applied for 1 hour in the dark. Cells were then washed and counterstained with DAPI (1:10,000 in 1X PBS) for 10 minutes to stain the nuclei. The labeled coverslips were mounted onto glass microscope slides using a drop of Dako Fluorescent Mounting Medium (Dako). Cells were visualized using a Zeiss Observer Z1 Inverted Microscope with appropriate filter settings.