Fluorescence Digital Image Gallery

Embryonic Swiss Mouse Fibroblast Cells (3T3)

In order to label the Golgi domain in mammalian cells with immunofluorescent probes, a candidate protein residing somewhere in the complex must be identified and targeted with a primary mono or polyclonal antibody. Giantin is a membrane-bound glycoprotein, which is resident in the cis and medial Golgi regions, and contains an elongated rod-like cytoplasmic domain that is at least 350 kiloDaltons in size. This highly conserved protein may be involved in intercisternal cross-bridge structures that are suspected to play a role in Golgi cisternae stacking. Polyclonal antibodies raised against the N-terminal 469 peptide residues of giantin react with a number of mammalian species, and monoclonal antibodies have been produced that detect the protein in avians, as well.

A semi-confluent culture of 3T3 cells (illustrated above) was fixed, permeabilized, and blocked with 10-percent normal goat serum in phosphate-buffered saline prior to immunofluorescent labeling with primary antibodies to giantin, a protein resident in the Golgi complex of mammalian cells. The culture was subsequently stained with a mixture of secondary antibodies conjugated to Alexa Fluor 488 in a mixture containing phalloidin conjugated to Alexa Fluor 568. The cell nuclei were counterstained with Hoechst 33342. Images were recorded in grayscale with a QImaging Retiga Fast-EXi camera system coupled to an Olympus BX-51 microscope equipped with bandpass emission fluorescence filter optical blocks provided by Omega Optical. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles.

View a larger image of the embryonic Swiss mouse fibroblast (3T3) cells.

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