Immunocytochemistry Protocol for Fluorescence Quantification

For immunocytochemistry, cells were fixed in 4%PFA for 10 minutes followed by 10 minutes in 0.3% triton in PBS. All antibodies except DDX3, which was made in our laboratory [31 (link)–33 (link), 37 (link), 38 (link)] is commercially available. Briefly, antibodies were diluted in 15% goat serum in DPBS (1:50 dilution) and incubated with tissue for 1 hr at room temperature. All antibodies were detected using fluorescently labeled secondary antibodies (1:200 dilution; Millipore) in 15% goat serum in DPBS for 1 hr at room temperature. Cells were counterstained with DAPI (Sigma) to detect nuclei. Negative controls were performed using secondary antibodies alone. For fluorescent quantitative analysis, images were captured using a Nikon E800 microscope and imported to Metamorph Imaging Software (Version 7.7). Total fluorescence units (FU) were calculated by dividing the number of pixels per area (3 areas measured per image), which are in arbitrary units. An adjacent area of the field that was not of interest was also measured to establish background fluorescence as previously described [44 (link)] At least, three independent experiments were performed for each treatment from which three separate 48-count tissue culture wells were counted per stain.