Mouse 3T3 fibroblasts stained for the nucleolar proteins nucleostemin (left panels, green) and fibrillarin (center, red). Top Row, control, center row 2h actinomycin D treatment, bottom row, 4h actinomycin D treatment. Panels at right show merge of nucleostemin and fibrillarin. Experiment illustrates the contrasting changes in intranuclear distribution of fibrillarin and nuceostemin in response to RNA synthesis inhibition.
Data were collected using a Leica DMIRB microscope equipped with a 100x objective (N.A. 1.4) and appropriate filter sets, and images captured using a Quantix 57 CCD camera (Roper Scientific Photometrics). For high resolution spatial mapping, three-dimensional optical stacks (containing 21 consecutive 0.25 micron slices) were captured using a PIFOC microscope focusing drive (Polytec PI). Images were dark current subtracted, and intensity scaled. See Fig 5 in Politz et al., 2005 Mol Biol Cell 16:3401-3410.
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