Immunofluorescence on isolated RLSMC. (A) α-Smooth muscle actin, a smooth muscle marker, shows stained stress fibers and smooth muscle actin. (B) Smooth-muscle-specific heavy Caldesmon stains positive. (C) Interstitial cells of cajal (ICC) marker, c-Kit, shows no positive areas, indicating the absence of ICC cells in the longitudinal smooth muscle culture. (D) Negative control of the rabbit secondary antibody indicates weak background staining like (C). Scale bar = 50 μm.