Techniques for Staining Specific Cellular Structures

Endoplasmic Reticulum

As with the Golgi apparatus (above), the best choice of probe for labeling the ER is probably an antibody against a resident protein, such as BiP, commercially available from many sources.

I have used 3,3'-dihexyloxacarbocyanine iodide, DiOC6(3) to label the ER in cultured cell lines. This dye possesses a positively charged polycyclic head domain and two hydrocarbon tails. It is cell-permeable, and readily accumulates in intracellular membranes. At low concentrations it accumulates in mitochondria, while at higher concentrations it labels the ER, which may be identified by its tubular morphology¹⁰. This dye is not fixable and is extracted by detergents, and so is not particularly useful for double-labeling with antibodies. Several laboratories have used it to study membrane dynamics in living cells, however. DiOC6(3) is a very bright dye, but is not very photostable, and I have found it useful to include N-propyl gallate in specimen preparations to retard photobleaching. The dye may be made as a 0.5 mg/mL stock in ethanol and is stored in the dark below 0°C. It may be diluted to 2.5 µg/mL into growth medium and used to stain living cells for 5 minutes. Alternatively, cells may be fixed with low (0.25%) concentrations of glutaraldehyde and then stained with 2.5 µg/mL diluted into PBS for 10 seconds. At higher concentrations, the dye begins to label cytosolic structures indiscriminately. Following labeling, samples should be mounted in PBS supplemented with 3% N-propyl gallate and imaged immediately. Cellular membranes will begin to bleb shortly thereafter, and the dye will redistribute.

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