Techniques for Staining Specific Cellular Structures

DNA

Fluorescent staining of the nuclei of cells is often useful in order to determine the status of the cell cycle of individual cells, or to provide a cytological 'landmark' for double staining, or as a counter-stain to delineate different populations of cells within tissues. There are literally dozens of DNA-specific fluorescent dyes to chose from, but I have used five of them predominantly, depending on the desired wavelength and microscope to be utilized. They are all intercalation agents, and only fluoresce when bound to nucleic acids.

The first two - DAPI and bisbenzimide (or Hoescht 33245) - are dyes that bind to DNA with very high specificity and photostability. They are cell permeable and readily cross the cell membrane. Both dyes are dissolved in de-ionized water at a concentration of 10 mg/mL to make a 1000X stock and are kept stored as aliquots below 0°C. They should be diluted into buffer and used to stain specimens for 10 to 30 minutes. Both dyes exhibit excitation wavelengths around 370 nm and emission peaks around 450 nm. These spectral characteristics make them particularly useful for triple labeling for fluorescence microscopy. [Note: the confocal microscope housed in the Beckman Institute is not currently equipped to excite dyes in the UV wavelengths.] Samples stained with these dyes should be documented with in one to two weeks after mounting as they will dissociate slowly and produce background autofluorescence in the hydrophobic conditions of some mounting media. The third is propidium iodide, which is a red dye (ex. 530 nm/em. 615 nm). PI is extremely photostable and is very robust even under the confocal microscope. It is prepared by dissolving in de-ionized water at a concentration of 10 mg/mL to make a 1000X stock and is stored as aliquots below 0°C. It should be diluted into buffer immediately prior to use and used to stain specimens for 10 to 30 minutes. PI will bind to RNA as well as DNA, so there is an additional step that must be performed when using this dye to stain nuclei. Following fixation and permeabilization, samples must be treated with RNAse (10 U/mL diluted into buffer for 30 minutes at 37°C in a humidified chamber) in order to avoid background staining of the cytoplasm. The fourth and fifth are green DNA-specific dyes - sytox green and chromomycin A3 (ex. 450 nm/em. 570 nm). Both are cell-impermeable, and so require that the specimens be fixed and permeabilized. Sytox green is available from Molecular Probes and comes as a solution in DMSO at 1000X concentration⁷. It may be diluted and used from this stock. This dye is very bright, but is not terribly photostable. Chromomycin A3 is available from numerous sources as a lyophilized powder. It should be reconstituted in buffer (i.e. PBS) to 10 mg/mL (for a 1000X stock) supplemented with 5 mM magnesium chloride. Chromomycin A3 must be complexed to magnesium in order to bind to DNA, so all staining solutions should be supplemented accordingly. This dye is fairly photostable, and is a good choice for a green nuclear stain for confocal microscopy.

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