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Human Fetal Lung Fibroblast Cells

Vimentin is a dynamic protein (58 kiloDaltons) that copolymerizes with other subunits to form intermediate filaments, which along with actin microfilaments and microtubules, comprise the cytoskeletal network. Vimentin is thought to play a key role in anchoring organelles within the cytosol and appears to be involved in communication and transport between the nucleus and cell surface.

Human Fetal Lung Fibroblast Cells

The log phase adherent culture of human lung cells (MRC-5 line) illustrated above was fixed and permeabilized with a cocktail of paraformaldehyde, Triton-X100, and glutaraldehyde before being blocked with 10-percent normal goat serum. Subsequent treatment with mouse anti-vimentin monoclonal primary antibodies was followed by goat anti-mouse secondary antibodies (IgG) conjugated to Cy3. In addition, filamentous actin was labeled with BODIPY FL conjugated to phallacidin and the intracellular mitochondrial network was visualized with MitoTracker Deep Red 633. Nuclei were counterstained with Hoechst 33258. Images were recorded in grayscale with a Hamamatsu ORCA AG camera system coupled to a ZEISS Axio Imager microscope equipped with bandpass emission fluorescence filter optical blocks provided by Chroma and Semrock. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles.