Comparison of fluorescence labeling of actin, vinculin, moesin and threonine558-phospho-moesin in NIH3T3 cells. Cells were rinsed with PBS(+) and fixed with TX100 (3.7% FA/0.2% TX100, see Table 1, A-D), TCA (see Table 1, E-H), or DOTMAC/PFA (see Table 1, I-L). Cells were double labeled with β-actin monoclonal antibodies (A5441) and a secondary antibody reagent conjugated to TRITC (A, E, and I), as well as with moesin polyclonal antibodies and secondary antibodies conjugated to FITC (C, G, and K). Vinculin and threonine558-phospho-moesin were also stained with vinculin monoclonal (B, F, and J) and threonine558-phospho-moesin polyclonal antibodies (D, H, and L), followed by FITC-conjugated anti-rabbit IgG. Note that the fluorescence intensity of each samples varied as summarized in Table 2. Images are shown at optimum fluorescence intensity. (M) Double exposure of J and TRITC-phalloidin-counterstained cells fixed with DOTMAC/PFA method. (N-M) Double exposure of I and K at different level of focus. Bars, 10 μm.